This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 95/11/1067    most recent 01.RES.0000149568.85071.FBv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nguyen, A. D. Articles by Fukai, T. Search for Related Content PubMed PubMed Citation Articles by Nguyen, A. D. Articles by Fukai, T. Related Collections Oxidant stress Mechanism of atherosclerosis/growth factors Pathophysiology

(Circulation Research. 2004;95:1067.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Fibulin-5 Is a Novel Binding Protein for Extracellular Superoxide Dismutase

Andrew D. Nguyen^*, Shinichi Itoh^*, Viktoria Jeney, Hiromi Yanagisawa, Mitsuaki Fujimoto, Masuko Ushio-Fukai, Tohru Fukai

From the Division of Cardiology (A.D.N., S.I., V.J., M.F., M.U.-F., T.F.), Department of Medicine, Emory University School of Medicine, Atlanta, Ga; and the Department of Molecular Biology (H.Y.), University of Texas Southwestern Medical Center, Dallas.

Correspondence to Tohru Fukai, MD, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, WMB 319, Atlanta, GA 30322. E-mail tfukai{at}emory.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The extracellular superoxide dismutase (ecSOD) plays an important role in atherosclerosis and endothelial function by modulating levels of the superoxide anion (O₂^·–) in the extracellular space. Although heparan sulfate proteoglycan is an important ligand for ecSOD, little is known about other biological binding partners of ecSOD. The goal of this study was to identify novel proteins that interact with ecSOD. A yeast two-hybrid screening of a human aorta cDNA library using ecSOD as bait identified fibulin-5 as a predominant binding protein for ecSOD. Further analysis showed that the binding domain of ecSOD within fibulin-5 mapped to its C-terminal domain. In vitro pulldown assays and coimmunoprecipitation analysis further confirmed that ecSOD interacts with fibulin-5 in vitro and in vivo. Studies using fibulin-5^–/– mice indicated that fibulin-5 is required for binding of ecSOD to vascular tissue. Importantly, the decrease in tissue-bound ecSOD levels in aortas from fibulin-5^–/– mice was associated with an increase in vascular O₂^·– levels. Furthermore, immunohistochemical analysis using ApoE^–/– mice suggested a codistribution of ecSOD and fibulin-5 in atherosclerotic vessels. In summary, we provide in this study the first evidence that the ecSOD-fibulin-5 interaction is required for ecSOD binding to vascular tissues, thereby regulating vascular O₂^·– levels. This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as atherosclerosis and hypertension in which oxidative stress is increased.

Key Words: superoxide dismutase • fibulin-5 • extracellular matrix • atherosclerosis • superoxide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular production of the superoxide anion (O₂^·–) is increased in many common cardiovascular diseases including atherosclerosis, hypercholesterolemia, hypertension, ischemic heart disease, diabetic cardiomyopathy, and heart failure.¹ One of the major cellular defenses against O₂^·– and formation of peroxynitrite is the superoxide dismutases (SODs).² In mammalian tissue, three isoforms of superoxide dismutase have been identified: Cu/ZnSOD, MnSOD, and extracellular superoxide dismutase (ecSOD). These isozymes differ in their location: Cu/ZnSOD is localized in the cytosol, MnSOD in the mitochondria, and ecSOD in the extracellular space. In the vessel wall, one-third to one-half of the total vascular SOD is ecSOD.³ In healthy vessels, ecSOD is produced predominately by vascular smooth muscle cells, but in atherosclerotic vessels, ecSOD is also generated by lipid-laden macrophages.^4,5 Because of its extracellular location, ecSOD plays an important role in modulating nitric oxide bioactivity by protecting nitric oxide from O₂^·– in the vascular extracellular space, especially in pathological states, such as atherosclerosis and hypertension where O₂^·– is increased.^2,6

The ecSOD is a secretory tetrameric glycoprotein with a heparin-binding domain.⁷ The protein is composed of an N-terminal signal peptide, which permits secretion from cell, an N-linked glycosylation site at Asn-89, which contributes to the solubility of the enzyme, an active site that binds copper and zinc, and a C-terminal region that corresponds to a heparin-binding domain.² Approximately 99% of the total ecSOD is tissue-bound, whereas a small proportion circulates in the blood.⁸ Heparan sulfate proteoglycan is a well-known ligand for ecSOD on cell surfaces and in the extracellular matrix.⁹ However, only a small portion of the tissue-bound ecSOD is displaced by heparin injection,⁸ suggesting that other ligands for ecSOD may exist.⁷

The major goal of this study was to identify novel proteins that interact with ecSOD. Using the yeast two-hybrid system, we discovered that fibulin-5 is an important biological ligand for ecSOD. Fibulin-5 is also known by the acronym EVEC (Embryonic Vascular EGF-like repeat Containing protein)¹⁰ and by the acronym DANCE (Developmental Arteries and Neural Crest EGF-like).¹¹ We further confirmed the interaction between ecSOD and fibulin-5 using in vitro pulldown assays and coimmunoprecipitation assays in mammalian cells as well as in insect cells. Furthermore, we found that ecSOD binds to the C-terminal domain of fibulin-5. Moreover, we examined functional significance of the interaction of ecSOD and fibulin-5 using fibulin-5^–/– mice, and found that vascular O₂^·– levels are markedly increased in fibulin-5^–/– mice in which ecSOD binding to tissue is markedly decreased.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals Studied
C57BL/6J mice and ApoE^–/– mice on a C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, Maine). The fibulin-5^–/– mice and control littermates were generated as previously described.¹²

Yeast Two-Hybrid Library Screening
To identify novel proteins that interact with ecSOD, we screened a human aorta cDNA library using ecSOD as bait and the MATCHMAKER GAL4 yeast two-hybrid system 3 (Clontech Laboratories Inc).

In Vitro Pulldown Assays and Generation of Recombinant ecSOD and Fibulin-5
For in vitro pulldown assays using recombinant ecSOD and fibulin-5, the human ecSOD and the human fibulin-5 were overexpressed in a Drosophila expression system (Invitrogen Corp). In vitro pulldown assays were performed as previously described.¹³

Coimmunoprecipitation of ecSOD and Fibulin-5 in Stably Transfected Drosophila Schneider Cells and CHO Cells
The Drosophila Schneider cells and CHO cells stably expressing ecSOD were generated according to the manufacturer’s instructions. Coimmunoprecipitation of ecSOD and fibulin-5 in those cells were performed as previously described.¹³

Immunohistochemical Studies
Immunohistochemical analysis for ecSOD and fibulin-5 were performed as previously described.⁴

Western Analysis of ecSOD and Fibulin-5 in Plasma and Aortas From Fibulin-5^–/– Mice and Control Littermates
The protein expression of ecSOD and fibulin-5 in plasma and aortas was determined by Western blotting analysis, as previously described elsewhere.⁴

Real-Time PCR
RNA was isolated and amplified as described previously with minor modifications.⁴ Primer sequences and cycling conditions are listed in expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org.

Dihydroethidium Labeling of Aortas From Fibulin-5^–/– Mice and Control Littermates
To measure ROS production in vessels in situ, frozen cross-sections of aortas were stained with dihydroethidium (Molecular Probes Inc) using a previously validated method.¹⁴

Materials
All chemicals and reagents were purchased from Sigma Chemical Company, unless otherwise specified.

Data Analysis
All data are expressed as mean±SEM. Comparisons between groups of animals or treatments were made by one-way ANOVA, followed by the Tukey-Kramer post hoc test. Values of P<0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fibulin-5 as a Novel Binding Protein for ecSOD
The yeast two-hybrid system was used to identify candidate proteins that interact with ecSOD. After an initial screening, 1257 independent clones grew as large colonies on the Trp^–/Leu^–/His^– plates. Of these, 389 clones exhibited ß-galactosidase activity, as tested by the filter assay. PCR was performed using the flanking primers specific for pACT2 plasmid to screen inserts ranging between 0.4 and 1.6 kb. Positive clones were sequenced, and DNA homology searches using the NCBI BLAST program identified six different partial clones of fibulin-5 (1 through 6) (Figure 1A). Those cDNAs were in frame with the GAL-4 activation domain of the pACT2 plasmid. Importantly, perlecan, one of major heparan sulfate proteoglycan in the vessel wall and an established physiological ligand for ecSOD,^8,15–18 was also included in our positive clones from the yeast two-hybrid screening, validating the yeast two-hybrid system in the present study.

View larger version (23K): [in this window] [in a new window]   Figure 1. Mapping the ecSOD binding site in fibulin-5 by the yeast two-hybrid system. A, Schematic representation of fibulin-5 and its various deletion mutants. Fibulin-5 has been subdivided as indicated at the top.10,11 Black boxes represent N-terminal signal sequences, and ovals represent calcium binding EGF-like (cbEGF) domains. An RGD motif is indicated in the first cbEGF domain. Position numbers of the amino acid residues are indicated. B, Semiquantitative assessment of growth in Trp–/Leu–/His– media (left), representative filter ß-galactosidase assays (center) and liquid luminescent ß-galactosidase assays (right) of yeast cells coexpressing ecSOD and full-length fibulin-5 or its several deletion mutants (1 through 7). Growth is designated by semiquantitative assessment with maximal growth at +++. The quantitative values are presented as mean±SD (n=5) and are expressed as relative light units (RLU) normalized on cell content (OD600). pGBKT7-Lam/pGAD-T and pGBKT7–53/pGAD-T were used as negative and positive controls, respectively.

To verify fibulin-5 as an ecSOD binding protein, we cloned the full coding sequence of fibulin-5 cDNA into the plasmids carrying either the GAL-4 DNA-binding domain (pGBKT7) or the GAL4 activation domain (pACT2) and adopted them as either prey or bait with ecSOD. As positive and negative controls, we used pGBKT7–53/pGADT7-T and pGBKT7-Lam/pGADT7-T, respectively. Figure 1B shows a robust growth of yeast coexpressing ecSOD and fibulin-5 (pGBKT7-ecSOD/pACT-fibulin-5) in Trp^–/Leu^–/His^– media. Furthermore, yeasts coexpressing ecSOD and fibulin-5 showed a marked production of blue colonies in filter ß-galactosidase assays and also showed a marked increase in ß-galactosidase activity in liquid ß-galactosidase assays (Figure 1B). These findings further confirmed the interaction of ecSOD with fibulin-5.

EcSOD Binds Specifically to the C-Terminal Domain of Fibulin-5
We next determined the ecSOD binding site in fibulin-5. As shown in Figure 1B, yeast coexpressing ecSOD and full-length fibulin-5 or one of its several deletion mutants (1 through 6) exhibited a robust growth in Trp^–/Leu^–/His^– media, a marked production of blue colonies in filter ß-galactosidase assays, and a marked increase in ß-galactosidase activity in liquid ß-galactosidase assays. In contrast, the fibulin-5 deletion mutant (7) lacking C-terminal domain (amino acids 320 to 448) showed neither growth nor ß-galactosidase activity. These results indicate that ecSOD interacts specifically with the C-terminal domain of fibulin-5 (amino acids 320 to 448).

Interaction of ecSOD and Fibulin-5 in Cell-Free Systems
To determine whether fibulin-5 directly interacts with ecSOD, we performed in vitro pulldown assays using recombinant ecSOD with V₅ tag (30 kDa) and recombinant fibulin-5 with Myc tag (66 kDa) proteins isolated and purified from the media of stably transfected Drosophila Schneider cells. As shown in Figure 2A, Myc-tagged fibulin-5 protein bound to V₅-tagged ecSOD protein, but not to IgG alone, suggesting that ecSOD directly interacts with fibulin-5 in vitro.

View larger version (56K): [in this window] [in a new window]   Figure 2. A, In vitro pulldown assays. Recombinant ecSOD with V5 tag and recombinant fibulin-5 with Myc tag proteins isolated and purified from the conditioned media of stably transfected Drosophila Schneider cells were coimmunoprecipitated (IP) as indicated, followed by immunoblotting (IB) with the anti-Myc antibody to detect fibulin-5 and the anti-V5 antibody to detect ecSOD. IgG was included as a negative control for the immunoprecipitation. Recombinant ecSOD with V5 tag and recombinant fibulin-5 with Myc tag proteins were included as a positive control. B and C, Coimmunoprecipitation of V5-tagged ecSOD and Myc-tagged fibulin-5 in stably transfected Drosophila Schneider cells (B) and stably transfected CHO cells (C). Two days after transient transfection with Myc-tagged fibulin-5, the conditioned media in Drosophila Schneider cells (B) or CHO cells (C) stably expressing ecSOD were immunoprecipitated (IP) with the antibody, as indicated followed by immunoblotting (IB) with the anti-Myc antibody to detect fibulin-5 and the anti-V5 antibody to detect ecSOD. Recombinant ecSOD with V5 tag and recombinant fibulin-5 with Myc tag proteins were included as a positive control. IgG was included as a negative control for the immunoprecipitation. D, Immunohistochemical analysis of fibulin-5 expression in ApoE–/– mouse aorta. Immunostaining was performed using an anti-rat fibulin-5 antibody (1:80) followed by a biotin-conjugated goat anti-rabbit IgG (1:100). Brown, Immunoreactivity with fibulin-5 is prominent in the endothelial surface and adventitia. Top panel shows low magnification, and bottom panel shows high magnification of atherosclerotic lesion. E, Immunohistochemical analysis of ecSOD expression in ApoE–/– mouse aorta. Immunostaining for ecSOD was performed using the anti-mouse ecSOD antibody (1:10 000) followed by a biotin-conjugated goat anti-rabbit IgG (1:100). Note that ecSOD and fibulin-5 are partially codistributed on the endothelial surface, in the extracellular matrix, and in the adventitia in ApoE–/– mouse aorta.

Coimmunoprecipitation of ecSOD and Fibulin-5 in Stably Transfected Cells
To confirm further the interaction of ecSOD and fibulin-5 in vivo, we performed coimmunoprecipitation assays in Drosophila Schneider 2 (Figure 2B) and CHO cells (Figure 2C) stably expressing ecSOD. These cells have been shown to allow proper glycosylation and secretion of a variety of mammalian proteins.^19,20 When Drosophila Schneider cells or CHO cells stably expressing V₅-tagged ecSOD were transiently transfected with Myc-tagged fibulin-5, ecSOD was coimmunoprecipitated with fibulin-5 in the conditioned media from these cells (Figure 2B and 2C). These data clearly suggest that both ecSOD and fibulin-5 interact in vivo.

Codistribution of ecSOD and Fibulin-5 in Control and Atherosclerotic Vessels
To determine whether ecSOD and fibulin-5 colocalize in intact vessels, we performed immunohistochemical analysis in control and atherosclerotic vessels. In control mouse aorta, ecSOD and fibulin-5 were codistributed in medial layer, whereas in atherosclerotic vessels from ApoE^–/– mice, they were codistributed on the endothelial cell surface, in the extracellular matrix, and in the adventitia (Figure 2D and 2E). These results suggest that ecSOD binds to fibulin-5 in vivo in both control and atherosclerotic vessels. Of note, fibulin-5 protein is abundantly expressed in normal adult vessels, whereas fibulin-5 mRNA expression is markedly less in aortas from control mice compared with those from ApoE^–/– mice (online Figure I).

Increase in Plasma ecSOD Level and Decrease in Tissue-Bound ecSOD Level in Fibulin-5^–/– Mice
To examine whether ecSOD binds to vascular tissue through interaction with fibulin-5 in intact vessels, we used fibulin-5^–/– mice and control littermates. Plasma ecSOD levels in fibulin-5^–/– mice were significantly increased by 2.4±0.2-fold as compared with levels in control littermates (Figure 3A). To test whether the increase in plasma ecSOD in fibulin-5^–/– mice was caused by a decrease in ecSOD binding to tissue, we next performed immunohistochemical analysis. EcSOD immunostaining was markedly decreased in aortas of fibulin-5^–/– mice compared with controls (Figure 3B). Consistent with this, Western analysis demonstrated that protein levels of ecSOD were decreased in aortas from fibulin-5^–/– mice (65±5%-fold decrease) compared with those from control littermates (Figure 3C). In contrast, protein levels of Cu/ZnSOD were not different between in aortas from fibulin-5^–/– and control mice (Figure 3C).

View larger version (60K): [in this window] [in a new window]   Figure 3. A, Western blotting analysis of plasma ecSOD in control littermates and fibulin-5–/– mice. Ten and twenty µg of protein from plasma of both control and fibulin-5–/– mice were loaded in adjacent lanes and size separated by SDS-PAGE. After transferring to a nitrocellulose membrane, ecSOD protein was detected by immunoblotting with a polyclonal antibody against ecSOD as previously described.4 *P<0.01 compared with wild-type aortas. B, Immunohistochemical analysis of ecSOD expression in aortas of control littermates and fibulin-5–/– mice. Immunostaining for ecSOD in aortas from control (A) and fibulin-5–/– (B) mice was performed by identical methods as described for Figure 4. Brown, Immunoreactivity with ecSOD. NC (negative control), Absence of staining in aortas from control (C) and fibulin-5–/– (D) mice when primary antibody was replaced with rabbit IgG. C, West-ern analysis of protein expression of ecSOD, Cu/ZnSOD, and fibulin-5 in aortas of control littermates and fibulin-5–/– mice. a, Representative Western blots for fibulin-5–/– and control littermates. Five and ten µg of protein from tissue homogenates of aortas of both fibulin-5–/– and control mice were loaded in adjacent lanes and size separated by SDS-PAGE. After transfer to a nitrocellulose membrane, fibulin-5, ecSOD, and Cu/ZnSOD proteins were detected by immunoblotting with their respective antibodies. b, Densitometric analysis of Western blots for ecSOD, Cu/ZnSOD, and fibulin-5 in control and fibulin-5–/– mice. Data are mean±SEM (n=3 for both groups). *P<0.01; #P<0.05 vs control cells.

To exclude the possibility that the decrease in ecSOD protein expression in aortas of fibulin-5^–/– mice was caused by a decrease in ecSOD mRNA, we performed real-time PCR. As shown in Figure 4A, ecSOD mRNA levels in aortas from fibulin-5^–/– mice and control littermates did not differ.

View larger version (56K): [in this window] [in a new window]   Figure 4. A, Real-time quantitative RT-PCR analysis to examine the ecSOD mRNA in aortas from control littermates and fibulin-5–/– mice. GAPDH mRNA levels were used as internal control. Mean data for three separate experiments. B, Immunohistochemical analysis of perlecan expression in aortas of control littermates and fibulin-5–/– mice. Immunostaining in aortas from control (A) and fibulin-5–/– (B) mice was performed using the anti-perlecan antibody (1:500), followed by HRP-conjugated goat anti-rat IgG (1:200). Brown, Immunoreactivity with perlecan. NC (negative control), Absence of staining in aortas from control (C) and fibulin-5–/– (D) mice when primary antibody was replaced with rat IgG.

It has been shown that heparan sulfate proteoglycan is an important ligand for ecSOD in the extracellular matrix.⁹ To eliminate the possibility that the decrease in ecSOD protein expression was caused by a decrease in the amount of heparan sulfate proteoglycan, we performed immunohistochemical analysis of perlecan, one of the major heparan sulfate proteoglycans in the vessel wall. Importantly, immunostaining of perlecan was not altered in aortas from fibulin-5^–/– mice compared with aortas from control mice (Figure 4B). Moreover, it has been shown that the C-terminal heparin-binding domain of ecSOD is cleaved during intracellular proteolytic processing,.^21,22 In this study, Western blots consistently revealed a full-length (upper band) and a proteolyzed (lower band) ecSOD in mouse aortas (Figure 3C).⁴ However, the ratio of proteolyzed (lacking heparin-binding domain) to nonproteolyzed ecSOD was not altered in aortas from fibulin-5^–/– mice compared with those from control mice (45±9% versus 54±13%, respectively) (Figure 3C). Taken together, these data suggest that the decrease in tissue-bound ecSOD in fibulin-5^–/– mice is caused by the loss of ecSOD binding to fibulin-5, and not caused by altered posttranslational processing in these animals.

Increased O₂^·– Production in Aortas From Fibulin-5^–/– Mice
To determine the functional significance of the decrease in tissue-bound ecSOD in fibulin-5^–/– mice, we examined O₂^·– production in aortas from fibulin-5^–/– mice and control littermates using the dihydroethidium (DHE) fluorescence method. DHE (2 µmol/L) is a fluorescent dye that has been shown to specifically detect O₂^·– in situ.¹⁴ DHE staining clearly demonstrated that superoxide production was markedly increased in aortas from fibulin-5^–/– mice as compared with those from control littermates (Figure 5A and 5B). Importantly, the fluorescence signal was markedly decreased by the addition of SOD, suggesting that DHE staining mainly reflects an increase in O₂^·–. Furthermore, additional experiments using recombinant ecSOD and fibulin-5 proteins showed that fibulin-5 had no direct effect on ecSOD activity (data not shown). Taken together, these findings suggest that fibulin-5 plays an important role in ecSOD binding to the tissue, thereby modulating vascular O₂^·– levels.

View larger version (16K): [in this window] [in a new window]   Figure 5. In situ detection of superoxide production with dihydroethidium (DHE) in aortas from control littermates and fibulin-5–/– mice. A, Fresh-frozen control (top left) and fibulin-5–/– (top right) aortas were incubated with DHE for 30 minutes. SOD (500 U/mL) applied topically to the sequential cut sections of control (bottom left) and fibulin-5–/– (bottom right) aortas during the 30 minute incubation with DHE. SOD abolished the fluorescence, confirming specificity of the fluorescent signal for O2–. Data are representative of 3 separate experiments. e indicates endothelium; m, media; a, adventitia. B, Digital scans of DHE-stained aortas from control and fibulin-5–/– mice were quantified using NIH Image software. Results shown are mean±SEM. *P<0.01 compared with control aortas.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, using a yeast two-hybrid system, we discovered fibulin-5 as a novel binding protein for ecSOD. The functional significance of this interaction was demonstrated by the observation that vascular O₂^·– levels are robustly increased in fibulin-5^–/– mice in which ecSOD binding to vascular tissue is markedly reduced. Moreover, we found a potential codistribution of ecSOD and fibulin-5 in both control and atherosclerotic vessels. Given that ecSOD plays an important role in scavenging O₂^·– in vascular extracellular space, ecSOD binding to fibulin-5 may represent a novel mechanism by which ecSOD regulates vascular redox state.

In vitro pulldown assays confirmed a direct interaction between ecSOD and fibulin-5. This interaction in vivo was further verified by coimmunoprecipitation of ecSOD and fibulin-5 in mammalian cells as well as in insect cells. Moreover, yeast two-hybrid mapping experiments identified that the binding site of ecSOD in fibulin-5 encompasses a globular cysteine-free C-terminal domain of fibulin-5, ie, residues 320 to 448. The C-terminal domain of fibulin-5 is a unique module for the fibulin family²³ and shares a significant homology with that of fibulin-3 and fibulin-4 (human fibulin-5 versus fibulin-4, 53%; human fibulin-5 versus fibulin-3, 50%; fibulin-3 versus fibulin-4, 53%), but a weak homology with that of fibulin-1C, fibulin-1D, and fibulin-2. Of interest, the C-terminal region of fibulin-5 physically interacts with lipoprotein(a) (Lp(a)). Elevated levels of Lp(a) have been recognized as an independent risk factor for atherosclerosis.²⁴ This region also physically interacts with lysyl oxidase-like 1 protein (LOXL1), a critical component for elastic fiber homeostasis.²⁵ Thus, it is possible that ecSOD may be involved in regulating interactions between fibulin-5 and either Lp(a) or LOXL1. Of note, fibulin-5 has been shown to bind to _vß₃, _vß₅, and ₉ß₁ integrins and to mediate endothelial cell adhesion via its RGD motif.²⁶ Moreover, it has been reported that integrin activities are regulated by extracellular redox state.^27,28 As such, it is tempting to speculate that ecSOD, a potent superoxide scavenger in the extracellular space, may participate in regulating function of integrins via binding to fibulin-5.

In the present study, we determined the interaction of ecSOD with fibulin-5 in vivo using fibulin-5^–/– mice (Figure 6). Immunohistochemical analysis demonstrates that ecSOD codistributes with fibulin-5 in medial layer of aorta from control mice (Figure 2D) and a marked decrease in ecSOD staining in that from fibulin-5^–/– mice (Figure 3B). In parallel, fibulin-5^–/– mice showed a significant increase in plasma ecSOD levels, and a marked decrease in tissue-bound ecSOD levels in aorta, compared with control mice in which fibulin-5 protein is abundantly expressed (Figure 3C). Of note, the ecSOD mRNA levels were not changed in aortas from fibulin-5^–/– mice as assessed by real-time PCR and fibulin-5^–/– mice showed neither a decrease in immunostaining of perlecan, which is one of the major components of heparan sulfate proteoglycans in the vessel wall nor an increase in heparin-binding domain cleaved ecSOD. Furthermore, numerous studies have demonstrated that affinity for heparan sulfate proteoglycan is important for localization of ecSOD in the extracellular matrix,^8,15–18 and recent reports indicate that enhanced proteolysis of the heparin-binding region of ecSOD significantly alters its tissue localization during pathological processes, such as lung injury.^21,22,29,30 In addition to our current findings, we have previously shown that the level of hydroxyproline, an indicator of collagen content, was not changed in fibulin-5^–/– mice,¹² although type I collagen is another ligand for ecSOD.³¹ Taken together, these findings strongly suggest that ecSOD binds to vascular extracellular matrix not only through the interaction with heparan sulfate proteoglycan but also with fibulin-5, which may explain why only a small portion (3%) of the tissue-bound ecSOD is displaced by heparin injection. Further studies will be required to investigate how the interaction of ecSOD and fibulin-5 are regulated and whether their interaction is observed in other tissues. However, because ecSOD protein expression is not completely abolished in aortas from fibulin-5^–/– mice, it is possible that ecSOD may bind to other ligands.

View larger version (17K): [in this window] [in a new window]   Figure 6. Proposed model illustrating the role of the interaction of ecSOD with fibulin-5 in the vessel wall. Left (control mice), Fibulin-5 binds to ecSOD via its C-terminal domain and binds to vascular integrins via its N-terminal RGD motif. Interaction of ecSOD with fibulin-5 plays an important role in the regulation of tissue-bound ecSOD levels in the vasculature. Moreover, fibulin-5 also binds to tropoelastin through its calcium-binding EGF repeat motif, which is essential for elastic fiber development.12,26 Right (fibulin-5–/– mice), EcSOD cannot bind to the extracellular matrix because of the absence of fibulin-5, resulting in decreased amounts of tissue-bound ecSOD and increased plasma ecSOD levels.

It has been shown that ecSOD plays an important role in regulating basal O₂^·– level in vascular tissue.⁶ We therefore measured vascular O₂^·– levels in control and fibulin-5^–/– mice to determine the functional significance of interaction of ecSOD and fibulin-5 in vivo. Figure 5 demonstrates that a marked decrease in tissue-bound ecSOD levels in fibulin-5^–/– mice is associated with an increase in vascular O₂^·– level assessed by SOD inhibitable dihydroethidium (DHE) fluorescence signal. We also found that recombinant fibulin-5 has no effect on ecSOD activity. Taken together, these results suggest an essential role of ecSOD binding to fibulin-5 in modulating basal levels of O₂^·– in vascular tissue. Because SOD cannot enter the intracellular space, the detected SOD inhibitable DHE signal may mainly reflect the O₂^·– derived from extracellular space. Indeed, oxyethidium, a specific fluorescent product by the reaction of dihydroethidium and superoxide anion,³² is cell-permeable (unpublished observation, 2004). However, we cannot exclude the possibility that an increase in vascular O₂^·– level in fibulin-5^–/– mice is caused by other mechanisms including enhanced superoxide generation system. This point requires further investigation.

To gain insight into the role of interaction of ecSOD with fibulin-5 in atherosclerosis, where O₂^·– is increased, we performed immunohistochemical analysis of ecSOD and fibulin-5 in atherosclerotic vessels from ApoE^–/– mice. We demonstrated that ecSOD partially codistributes with fibulin-5 in endothelial surface, extracellular matrix, and adventitia (Figure 2E), which is different from their codistribution in medial layer of aorta observed in control mice. Of interest, previous studies have shown that both ecSOD^4,33 and fibulin-5^10,11 are highly induced in balloon-injured arteries and in atherosclerosis. Thus, it is possible that an increase in protein expression of fibulin-5 enhances binding of ecSOD to vascular tissue, resulting in increased vascular ecSOD protein expression. Taken together, these findings most likely represent a novel feed forward protective mechanism whereby ecSOD modulates vascular O₂^·– levels through interaction with fibulin-5.

Of note, the present study shows that fibulin-5 protein is abundantly expressed in normal adult vessels, although original reports demonstrated that fibulin-5 mRNA is very low in normal adult cells, and is markedly increased after vascular injury or in atherosclerosis.^10,11 Our results also show that fibulin-5 mRNA is markedly less expressed in aortas from control mice compared with those from ApoE^–/– mice (online Figure I). Because fibulin-5 is an elastin binding protein as well as a secretory protein, it is possible that fibulin-5 may be accumulated on the elastic fibers after secretion, which may contribute to increased protein expression in aorta.

Several lines of evidence suggest that ecSOD plays an important role in regulating blood pressure. Jung et al⁶ reported that ecSOD deficiency enhanced an increase in blood pressure and O₂^·– in response to angiotensin II and in the two-kidney and one-clip model. Furthermore, Chu et al³⁴ showed that gene transfer of ecSOD reduces arterial pressure in a genetic model of hypertension. Of interest, fibulin-5^–/– mice, in which tissue-bound ecSOD was markedly decreased in aorta, also show an increase in systolic blood pressure, pulse pressure, and aortic stiffness.^12,26 In addition, it has been shown that aortic stiffness, which contributes to an increase in pulse pressure, is positively associated with oxidative stress³⁵ through increasing elastin degradation,³⁶ elastase activity via activation of increased MMP,³⁷ and phenotypic modulation of medial vascular smooth muscle cells from the contractile type to the synthetic one.³⁵ Thus, it is conceivable that fibulin-5 may also participate in regulating aortic stiffness by modulating vascular redox state through binding to ecSOD, thereby controlling pulse pressure and aortic compliance.

In summary, we have demonstrated that the interaction of ecSOD with fibulin-5 is essential to ecSOD binding to vascular tissue, which modulates O₂^·– levels in the vasculature. This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as hypertension and atherosclerosis in which oxidative stress is highly elevated.

	Acknowledgments

 
This research was supported by NIH R01 HL70187, Program Project Grant HL58000, AHA National Scientist Development Grant 0030180N, and AHA Grant In Aid 0455242B. We thank Dr David G. Harrison for helpful discussions and Suzanne Mertens and Shelby Hacker for excellent technical assistance.

	Footnotes

 
*Both authors contributed equally to this study.

Original received July 12, 2004; revision received October 4, 2004; accepted October 25, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury, part II: animal and human studies. Circulation. 2003; 108: 2034–2040.[Free Full Text]
2. Fukai T, Folz RJ, Landmesser U, Harrison DG. Extracellular superoxide dismutase and cardiovascular disease. Cardiovasc Res. 2002; 55: 239–249.[Abstract/Free Full Text]
3. Strålin P, Karlsson K, Johansson BO, Markland SL. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol. 1995; 15: 2032–2036.[Abstract/Free Full Text]
4. Fukai T, Galis ZS, Meng XP, Parthasarathy S, Harrison DG. Vascular expression of extracellular superoxide dismutase in atherosclerosis. J Clin Invest. 1998; 101: 2101–2111.[Medline] [Order article via Infotrieve]
5. Luoma JS, Stralin P, Marklund SL, Hiltunen TP, Sarkioja T, Yla-Herttuala S. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler Thromb Vasc Biol. 1998; 18: 157–167.[Abstract/Free Full Text]
6. Jung O, Marklund SL, Geiger H, Pedrazzini T, Busse R, Brandes RP. Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice. Circ Res. 2003; 93: 622–629.[Abstract/Free Full Text]
7. Oury TD, Crapo JD, Valnickova Z, Enghild JJ. Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase. Biochem J. 1996; 317: 51–57.[Medline] [Order article via Infotrieve]
8. Karlsson K, Marklund SL. Heparin-induced release of extracellular superoxide dismutase to human blood plasma. Biochem J. 1987; 242: 55–59.[Medline] [Order article via Infotrieve]
9. Fattman CL, Schaefer LM, Oury TD. Extracellular superoxide dismutase in biology and medicine. Free Radic Biol Med. 2003; 35: 236–256.[CrossRef][Medline] [Order article via Infotrieve]
10. Kowal RC, Richardson JA, Miano JM, Olson EN. EVEC, a novel epidermal growth factor-like repeat-containing protein upregulated in embryonic and diseased adult vasculature. Circ Res. 1999; 84: 1166–1176.[Abstract/Free Full Text]
11. Nakamura T, Ruiz-Lozano P, Lindner V, Yabe D, Taniwaki M, Furukawa Y, Kobuke K, Tashiro K, Lu Z, Andon NL, Schaub R, Matsumori A, Sasayama S, Chien KR, Honjo T. DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries. J Biol Chem. 1999; 274: 22476–22483.[Abstract/Free Full Text]
12. Yanagisawa H, Davis EC, Starcher BC, Ouchi T, Yanagisawa M, Richardson JA, Olson EN. Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo. Nature. 2002; 415: 168–171.[CrossRef][Medline] [Order article via Infotrieve]
13. Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M, Quinn MT, Pagano PJ, Johnson C, Alexander RW. Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res. 2002; 91: 1160–1167.[Abstract/Free Full Text]
14. Miller FJ, Jr., Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.[Abstract/Free Full Text]
15. Karlsson K, Marklund SL. Extracellular superoxide dismutase in the vascular system of mammals. Biochem J. 1988; 255: 223–228.[Medline] [Order article via Infotrieve]
16. Karlsson K, Lindahl U, Marklund SL. Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans. Biochem J. 1988; 256: 29–33.[Medline] [Order article via Infotrieve]
17. Karlsson K, Marklund SL. Binding of human extracellular-superoxide dismutase C to cultured cell lines and to blood cells. Lab Invest. 1989; 60: 659–666.[Medline] [Order article via Infotrieve]
18. Karlsson K, Sandstrom J, Edlund A, Edlund T, Marklund SL. Pharmacokinetics of extracellular-superoxide dismutase in the vascular system. Free Radic Biol Med. 1993; 14: 185–190.[CrossRef][Medline] [Order article via Infotrieve]
19. Grossmann M, Szkudlinski MW, Tropea JE, Bishop LA, Thotakura NR, Schofield PR, Weintraub BD. Expression of human thyrotropin in cell lines with different glycosylation patterns combined with mutagenesis of specific glycosylation sites. Characterization of a novel role for the oligosaccharides in the in vitro and in vivo bioactivity. J Biol Chem. 1995; 270: 29378–29385.[Abstract/Free Full Text]
20. Culp JS, Johansen H, Hellmig B, Beck J, Matthews TJ, Delers A, Rosenberg M. Regulated expression allows high level production and secretion of HIV-1 gp120 envelope glycoprotein in Drosophila Schneider cells. Biotechnology (NY). 1991; 9: 173–177.
21. Enghild JJ, Thogersen IB, Oury TD, Valnickova Z, Hojrup P, Crapo JD. The heparin-binding domain of extracellular superoxide dismutase is proteolytically processed intracellularly during biosynthesis. J Biol Chem. 1999; 274: 14818–14822.[Abstract/Free Full Text]
22. Bowler RP, Nicks M, Olsen DA, Thogersen IB, Valnickova Z, Hojrup P, Franzusoff A, Enghild JJ, Crapo JD. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase. J Biol Chem. 2002; 277: 16505–16511.[Abstract/Free Full Text]
23. Argraves WS, Greene LM, Cooley MA, Gallagher WM. Fibulins: physiological and disease perspectives. EMBO Rep. 2003; 4: 1127–1131.[CrossRef][Medline] [Order article via Infotrieve]
24. Kapetanopoulos A, Fresser F, Millonig G, Shaul Y, Baier G, Utermann G. Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain. Mol Genet Genomics. 2002; 267: 440–446.[CrossRef][Medline] [Order article via Infotrieve]
25. Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, Spencer JA, Yanagisawa H, Zuo J, Li T. Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet. 2004; 36: 178–182.[CrossRef][Medline] [Order article via Infotrieve]
26. Nakamura T, Lozano PR, Ikeda Y, Iwanaga Y, Hinek A, Minamisawa S, Cheng CF, Kobuke K, Dalton N, Takada Y, Tashiro K, Ross Jr J, Honjo T, Chien KR. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature. 2002; 415: 171–175.[CrossRef][Medline] [Order article via Infotrieve]
27. Yan B, Smith JW. A redox site involved in integrin activation. J Biol Chem. 2000; 275: 39964–39972.[Abstract/Free Full Text]
28. Smith JW, Yan B, Landry F, Lombardo CR. Redox control of integrin adhesion receptors. Methods Enzymol. 2002; 353: 156–163.[Medline] [Order article via Infotrieve]
29. Nozik-Grayck E, Dieterle CS, Piantadosi CA, Enghild JJ, Oury TD. Secretion of extracellular superoxide dismutase in neonatal lungs. Am J Physiol Lung Cell Mol Physiol. 2000; 279: L977–L984.[Abstract/Free Full Text]
30. Fattman CL, Chu CT, Kulich SM, Enghild JJ, Oury TD. Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment. Free Radic Biol Med. 2001; 31: 1198–1207.[CrossRef][Medline] [Order article via Infotrieve]
31. Petersen SV, Oury TD, Ostergaard L, Valnickova Z, Wegrzyn J, Thogersen IB, Jacobsen C, Bowler RP, Fattman CL, Crapo JD, Enghild JJ. Extracellular superoxide dismutase (EC-SOD) binds to type I collagen and protects against oxidative fragmentation. J Biol Chem. 2004; 279: 13705–13710.[Abstract/Free Full Text]
32. Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, Kalyanaraman B. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med. 2003; 34: 1359–1368.[CrossRef][Medline] [Order article via Infotrieve]
33. Leite PF, Danilovic A, Moriel P, Dantas K, Marklund S, Dantas AP, Laurindo FR. Sustained decrease in superoxide dismutase activity underlies constrictive remodeling after balloon injury in rabbits. Arterioscler Thromb Vasc Biol. 2003; 23: 2197–2202.[Abstract/Free Full Text]
34. Chu Y, Iida S, Lund DD, Weiss RM, DiBona GF, Watanabe Y, Faraci FM, Heistad DD. Gene transfer of extracellular superoxide dismutase reduces arterial pressure in spontaneously hypertensive rats: role of heparin-binding domain. Circ Res. 2003; 92: 461–468.[Abstract/Free Full Text]
35. Itoh S, Umemoto S, Hiromoto M, Toma Y, Tomochika Y, Aoyagi S, Tanaka M, Fujii T, Matsuzaki M. Importance of NAD(P)H oxidase-mediated oxidative stress and contractile type smooth muscle myosin heavy chain SM2 at the early stage of atherosclerosis. Circulation. 2002; 105: 2288–2295.[Abstract/Free Full Text]
36. Hayashi A, Ryu A, Suzuki T, Kawada A, Tajima S. In vitro degradation of tropoelastin by reactive oxygen species. Arch Dermatol Res. 1998; 290: 497–500.[CrossRef][Medline] [Order article via Infotrieve]
37. Rajagopalan S, Meng X-P, Ramasamy S, Harrison DG, Galis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro: Implications for atherosclerotic plaque stability. J Clin Invest. 1996; 98: 2572–2579.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				H. Dihazi, M. J. Koziolek, T. Sollner, E. Kahler, R. Klingel, R. Neuhoff, F. Strutz, and G. A. Mueller Protein adsorption during LDL-apheresis: proteomic analysis Nephrol. Dial. Transplant., April 8, 2008; (2008) gfn127v1. [Abstract] [Full Text] [PDF]

				M. C. Gongora and D. G. Harrison Sad Heart From No SOD Hypertension, January 1, 2008; 51(1): 28 - 30. [Full Text] [PDF]

				H. W. Kim, A. Lin, R. E. Guldberg, M. Ushio-Fukai, and T. Fukai Essential Role of Extracellular SOD in Reparative Neovascularization Induced by Hindlimb Ischemia Circ. Res., August 17, 2007; 101(4): 409 - 419. [Abstract] [Full Text] [PDF]

				M. Hirai, T. Ohbayashi, M. Horiguchi, K. Okawa, A. Hagiwara, K. R. Chien, T. Kita, and T. Nakamura Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo J. Cell Biol., March 26, 2007; 176(7): 1061 - 1071. [Abstract] [Full Text] [PDF]

				T. Fukai Extracellular SOD Inactivation in High-Volume Hypertension: Role of Hydrogen Peroxide Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 442 - 444. [Full Text] [PDF]

				O. Jung, S. L. Marklund, N. Xia, R. Busse, and R. P. Brandes Inactivation of Extracellular Superoxide Dismutase Contributes to the Development of High-Volume Hypertension Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 470 - 477. [Abstract] [Full Text] [PDF]

				P. G. Drewes, H. Yanagisawa, B. Starcher, I. Hornstra, K. Csiszar, S. I. Marinis, P. Keller, and R. A. Word Pelvic Organ Prolapse in Fibulin-5 Knockout Mice: Pregnancy-Induced Changes in Elastic Fiber Homeostasis in Mouse Vagina Am. J. Pathol., February 1, 2007; 170(2): 578 - 589. [Abstract] [Full Text] [PDF]

				P.-P. Kuang, M. Joyce-Brady, X.-H. Zhang, J.-C. Jean, and R. H. Goldstein Fibulin-5 gene expression in human lung fibroblasts is regulated by TGF-beta and phosphatidylinositol 3-kinase activity Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1412 - C1421. [Abstract] [Full Text] [PDF]

				N. V. Lee, J. C. Rodriguez-Manzaneque, S. N.-M. Thai, W. O. Twal, A. Luque, K. M. Lyons, W. S. Argraves, and M. L. Iruela-Arispe Fibulin-1 Acts as a Cofactor for the Matrix Metalloprotease ADAMTS-1 J. Biol. Chem., October 14, 2005; 280(41): 34796 - 34804. [Abstract] [Full Text] [PDF]

				C. S. Wilcox Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2005; 289(4): R913 - R935. [Abstract] [Full Text] [PDF]

				P. Nyberg, L. Xie, and R. Kalluri Endogenous Inhibitors of Angiogenesis Cancer Res., May 15, 2005; 65(10): 3967 - 3979. [Abstract] [Full Text] [PDF]

				V. Jeney, S. Itoh, M. Wendt, Q. Gradek, M. Ushio-Fukai, D. G. Harrison, and T. Fukai Role of Antioxidant-1 in Extracellular Superoxide Dismutase Function and Expression Circ. Res., April 15, 2005; 96(7): 723 - 729. [Abstract] [Full Text] [PDF]

				J. A. Spencer, S. L. Hacker, E. C. Davis, R. P. Mecham, R. H. Knutsen, D. Y. Li, R. D. Gerard, J. A. Richardson, E. N. Olson, and H. Yanagisawa Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration PNAS, February 22, 2005; 102(8): 2946 - 2951. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 95/11/1067    most recent 01.RES.0000149568.85071.FBv1