HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 28, 20959-20962, July 14, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/28/20959    most recent C000290200v1 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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malyankar, U. M. Articles by Giachelli, C. M. Search for Related Content PubMed PubMed Citation Articles by Malyankar, U. M. Articles by Giachelli, C. M. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Osteoprotegerin Is an _v3-induced, NF-B-dependent Survival Factor for Endothelial Cells^*

Uriel M. Malyankar, Marta Scatena, Katherine L. Suchland, Theodore J. Yun^§, Edward A. Clark^§, and Cecilia M. Giachelli^¶

From the Departments of  Bioengineering and ^§ Immunology, University of Washington, Seattle, Washington 98195

Received for publication, April 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Osteopontin protects endothelial cells from apoptosis induced by growth factor withdrawal. This interaction is mediated by the _v3 integrin and is NF-B-dependent (Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093). In the present study we used differential cloning to identify osteopontin-induced, NF-B-dependent genes in endothelial cells. One of the genes identified in this screen was osteoprotegerin, a member of the tumor necrosis factor receptor superfamily. By Northern and Western blot analysis, osteoprotegerin mRNA and protein levels were very low in endothelial cells plated on the non-integrin cell attachment factor, poly-D-lysine. In contrast, osteoprotegerin mRNA and protein levels were induced 5-7-fold following _v3 ligation by osteopontin. Osteoprotegerin induction by osteopontin was time-dependent and observed as early as 3 h following treatment. NF-B inactivation achieved by over expression of an IB super repressor in endothelial cells completely inhibited osteoprotegerin induction by osteopontin. Finally, purified osteoprotegerin protected endothelial cells with inactive NF-B from apoptosis induced by growth factor deprivation. These data suggest that _v3-mediated endothelial survival depends on osteoprotegerin induction by NF-B and indicate a new function for osteoprotegerin in endothelial cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Angiogenesis, the formation of capillaries from pre-existing blood vessels, occurs as a result of various normal and pathological processes, including ovulation, wound healing, and ischemic disease. The angiogenic process requires endothelial cells to acquire an altered phenotype, modify cell-cell contacts, migrate, proliferate, and re-establish cell-cell contacts to form patent tubes or endothelial sheets. Finally, mechanisms that control microvessel regression balance pro-angiogenic processes to regulate the extent, magnitude, and duration of angiogenesis.

It has become apparent that up-regulation of mechanisms that promote endothelial survival are essential during angiogenesis. This requirement was underscored by studies showing that inhibitors of _v3 integrin (but not 1 integrins) block angiogenesis by inducing apoptosis of migrating endothelial cells (1). While the precise mechanism by which blockade of _v3 induces apoptosis of endothelial cells is not clear, previous studies suggest that _v3 ligation modulates expression of apoptosis-regulatory genes (2, 3). For example, _v3 blockade caused activation of p53 and down-regulation of bcl-2 in endothelial cells isolated from CAM (2). In addition, previous studies implicate _v3-mediated activation of NF-B as an important endothelial cell survival pathway (4). The transcription factor NF-B is a pleiotropic regulator of many genes involved in immune and inflammatory responses. The NF-B family of proteins consists of homo- or heterodimeric subunits of the Rel family. In unstimulated cells, NF-B is localized in the cytoplasm in complex with an inhibitory protein, IB (5). Upon stimulation, the inhibitory IB becomes phosphorylated, ubiquitinated, and subsequently degraded by the proteosome machinery (6). This allows NF-B to translocate to the nucleus, bind DNA, and transactivate transcription of specific genes. In endothelial cells, apoptosis induced by growth factor deprivation was blocked by osteopontin, an _v3 ligand (4). The protective effect of osteopontin required nuclear translocation of the transcription factor, NF-B, since overexpression of a nondegradable form of IB (super repressor) blocked the effect.

Several recent studies have implicated NF-B as an important cell survival factor (4, 7-9). It has been hypothesized that NF-B-induced transcription of anti-apoptotic genes is responsible for its protective activity. In support of this, NF-B has been shown to up-regulate transcription of antiapoptotic molecules such as A1, A20, c-IAP1, c-IAP2, XIAP, TRAF2, and MnSOD (10-13). To determine whether these or unique factors might be responsible for the protective effects of NF-B in endothelial cells, we used suppressive subtraction hybridization to isolate endothelial genes that required active NF-B for induction by osteopontin. Here we report that one of the clones isolated in this screen was osteoprotegerin, a soluble member of the TNF¹ receptor superfamily and the recently discovered osteoclast differentiation inhibitory factor. We show that adding recombinant osteoprotegerin to endothelial cells prevents apoptosis due to serum withdrawal and NF-B blockade. These studies are the first to describe endothelial cell expression of osteoprotegerin and its potential role in endothelial cell survival mediated by integrin ligation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cell Culture-- Rat aortic endothelial cells (RAECs) and RAECN2 were passaged in MCDB 131 and RPMI 1640 medium, respectively, as described previously (4). RAECN2 cells express a nondegradable form of IB (IB super repressor) under the control of the zinc-inducible metallothionein promoter. We previously determined that translocation of NF-B to the nucleus in RAECN2 cells was inhibited when these cells were treated with 50 µM ZnSO₄ (4).

Construction of Subtracted Libraries-- A subtracted library of cDNAs overexpressed by endothelial cells plated on osteopontin and dependent on NF-B activation was made using the PCR-Select kit from CLONTECH (Palo Alto, CA). In brief, poly(A)⁺ RNA was prepared from RAECN2 cells that were plated on osteopontin in the presence or absence of 50 µM ZnSO₄ for 4 h using the Invitrogen FAStrac poly(A)⁺ prep kit (Invitrogen, Carlsbad, CA). In the presence of zinc, NF-B activity is blocked due to activation of the metallothionein promoter that drives expression of the dominant IB super repressor (4). One µg of RNA from each of the samples was used to prepare a population of subtracted cDNA clones from the cells grown on osteopontin in the absence of ZnSO₄ (NF-B active) using the manufacturer's protocol (CLONTECH, Palo Alto, CA). Osteopontin-induced, NF-B-dependent clones were subcloned into pCR2.1 using the T-A cloning kit (Invitrogen, Carlsbad, CA). The ligation mixture was transfected into SURE cells (Stratagene, La Jolla, CA). Single colonies were picked and screened for inserts using PCR. Randomly picked clones were sequenced using the T7 primer in pCR2.1 and sequences submitted for BLAST analysis using public domain NCBI data bases for nonredundant sequences and expressed sequence tags.

RNA Purification and Northern Blots-- Total RNA was isolated from confluent RAEC and RAECN2 cells and analyzed as described previously (14). Northern blot analysis was carried out by electrophoretic separation of 10 µg of total RNA using formaldehyde-agarose gels and subsequent transfer. Equal loading of RNA in the lanes was confirmed by examination of the membrane after staining with methylene blue. Relative levels of RNA were determined using the phosphorimager analysis facility in the Department of Pathology at the University of Washington. For Northern blot analyses, cDNA inserts were labeled using the Multiprime kit (Amersham Pharmacia Biotech) and [-³²P]dCTP. RNA levels were normalized using a radiolabeled glyceraldehyde-3-phosphate dehydrogenase probe as described previously (14). Radiolabeled oligonucleotides were purified using G-25 spin columns (Amersham Pharmacia Biotech).

Western Blots-- Equal volumes of media were concentrated using Microcon concentrators mixed with sample buffer, denatured by boiling, and applied to a 10% SDS-polyacrylamide gel. Identical gels were stained with Coomassie Blue (ISS Corp.) or transferred to polyvinylidene difluoride membrane. Western blot analyses were carried out using the FDCR-1 rabbit polyclonal antibody from Dr. E. Clark (15), HRP-labeled goat anti-rabbit secondary antibody (Jackson Laboratories, Bar Harbor, ME), an HRP detection system (Pierce), and a Nucleotech chemiluminescense imaging system.

Cell Survival Assay-- RAECN2 cells were plated on permanox slides that were coated with different substrates as described previously (4). Nuclei were stained with Hoechst 33342 dye (Molecular Bioprobes, Eugene, OR). Condensed and fragmented nuclei were scored as apoptotic. In the studies involving inhibition of apoptosis recombinant osteoprotegerin (R & D Systems, Minneapolis, MN) was added to the cells.

FACS Analysis-- RAECN2 cells were plated on osteopontin for 16 h in the presence and absence of ZnSO₄ as described earlier. Cells were washed with phosphate-buffered saline, removed mechanically, incubated in FACS medium (RPMI 1640 containing 2% FCS and 0.1% sodium azide) and treated with a FDCR-1 antiserum or preimmune serum at a dilution of 1:1000 at 4 °C for 1 h. The cells were washed, fixed using 1% paraformaldehyde, and incubated with fluorescein isothiocyanate-conjugated goat anti rabbit antibody (Amersham Pharmacia Biotech) at 4 °C for 30 min and analyzed by flow cytometry (15).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Isolation of _v3 Integrin-induced, NF-B-dependent Genes-- We previously demonstrated that growth factor-deprived endothelial cells undergo apoptosis when integrin-mediated adhesion is blocked. We have also shown that in cells plated on osteopontin, cell survival is mediated by _v3 integrin and dependent on translocation of NF-B to the nucleus (4). We have, as part of our previous studies, prepared a cell line RAECN2 that overexpresses a nondegradable form of IB (super repressor) and is regulated by the metallothionein promoter. Using these cells, we showed that translocation of NF-B to the nucleus and activity in RAECN2 cells was inhibited following treatment with 50 µM ZnSO₄ (4), because the super repressor IB cannot be phosphorylated and thus is not proteolytically degraded.

To find genes that play a role in endothelial cell survival, we created a subtraction library of clones from RAECN2 cells that were plated on osteopontin in serum-free medium and had active NF-B. These cells undergo apoptosis when ZnSO₄ is added to the media, thereby inactivating NF-B (Fig. 1A). cDNAs were prepared from cells subjected to these two different conditions and used in a suppressive subtraction hybridization technique (Fig. 1B). The subtracted inserts from the population of cells containing active NF-B were cloned into a plasmid vector, pCR2.1. Random clones were picked from the subtracted library, sequenced, and used for BLAST analysis using the NCBI data bases to identify the clones. The clones were tabulated depending on function (Table I), and their expression levels in apoptotic and non-apoptotic cells were analyzed by Northern blotting. As seen from this table, a number of the genes isolated (VCAM-1, iNOS, etc.) have been shown previously to be regulated by NF-B and involved in endothelial inflammatory processes (16). Of interest, one of the genes that we isolated in this screen was osteoprotegerin, which is required for osteoclast differentiation and blocks TRAIL-mediated apoptosis (17, 18). Expression of osteoprotegerin in endothelial cell has not been previously demonstrated therefore expression and function of osteoprotegerin in endothelial cells was investigated.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A, RAECN2 cells survive serum withdrawal-induced cell death when plated on osteopontin. In contrast, RAECN2 cells undergo apoptosis when NF-B is inactivated by the expression of an IB super repressor (ZnSO4). B, schematic representation of suppressive subtraction hybridization strategy used to isolate v3-induced, NF-B-dependent endothelial genes in this study.

View this table: [in this window] [in a new window]   Table I v3-induced, NF-B-dependent endothelial cell genes

Expression of Osteoprotegerin in Endothelial Cells-- We isolated total RNA from RAECN2 cells that were plated in serum-free medium on osteopontin for 20 h in the presence or absence of ZnSO₄ and used this RNA in a Northern blot. As shown in Fig. 2A, cells plated on osteopontin in the absence of zinc (NF-B active) express a high level of osteoprotegerin mRNA, while cells plated in the presence of zinc (NF-B-inactive) do not express osteoprotegerin (Fig. 2A). Expression of osteoprotegerin mRNA was time-dependent following attachment of cells to osteopontin (Fig. 2B).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Osteopontin adherent cells containing active NF-B express osteoprotegerin. A, total RNA from RAECN2 cells plated on osteopontin in the presence or absence of ZnSO4in serum-free medium was probed for osteoprotegerin. B, time course of osteoprotegerin mRNA expression in RAECN2 cells plated on osteopontin for the indicated times. C, Western blotting of medium from RAECN2 cells that were plated on osteopontin and PDL in the presence or absence of ZnSO4 using antiserum directed against osteoprotegerin shows expression of osteoprotegerin by cells with active NF-B. Position of migration of purified recombinant human OPG is shown (hrOPG). D, flow cytometry analysis of RAECN2 cells using anti-osteoprotegerin antisera shows cell-associated osteoprotegerin expression by RAECN2 cells (open peak on right) compared with negative staining by normal rabbit sera (filled peak on left).

To determine whether osteoprotegerin was secreted by these cells, we performed Western blot analysis on culture supernatants. As shown in Fig. 2C, the level of osteoprotegerin protein in conditioned medium from cells plated in serum free medium on osteopontin for 20 h was much greater than that seen in medium from cells plated in the presence of zinc. In contrast, very little osteoprotegerin protein was observed in RAECN2 cells plated on PDL. Thus, osteoprotegerin appears to be secreted in response to osteopontin treatment in RAEC.

Osteoprotegerin does not contain a transmembrane domain (17, 19); however, it has been reported to be associated with the cell surface in some cell types (15). We performed flow cytometry to examine if osteoprotegerin is associated with the cell surface. RAECN2 cells were plated on osteopontin and used for flow cytometry (Fig. 2D). The antiserum directed against osteoprotegerin showed a distinct increase in fluorescence compared with normal rabbit serum. Thus, at least some of the osteoprotegerin synthesized by endothelial cells may be associated with the surface of endothelial cells, possibly bound to some extracellular matrix protein expressed by RAECN2 cells.

Role of Osteoprotegerin in Endothelial Cell Survival-- The expression pattern of osteoprotegerin suggested that it might mediate the NF-B-dependent protective effects of _v3 ligation in endothelial cells. To examine this directly, we carried out cell survival studies with purified osteoprotegerin. As demonstrated previously, RAECN2 cells plated on osteopontin in the absence of ZnSO₄ (active NF-B) showed a low percentage of apoptotic nuclei. However, upon addition of 50 µM ZnSO₄, NF-B is inactivated, and serum deprivation now stimulates apoptosis (Fig. 1A). Addition of osteoprotegerin to the cell culture medium did not affect cells with active NF-B but almost completely blocked apoptosis of cells with inactive NF-B (Fig. 3). Osteoprotegerin promoted a dose-dependent decrease in the percentage of apoptotic nuclei (Fig. 3), with half-maximal effects between 1 and 2 µg/ml osteoprotegerin. Addition of 5 µg/ml of exogenous osteoprotegerin decreased the level of apoptotic nuclei about 5-fold and almost to control levels. These data implicate osteoprotegerin as a potent survival factor for endothelial cells in response to serum deprivation.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Osteoprotegerin prevents apoptosis in RAECN2 cells in a dose-dependent manner. Increasing concentrations of human recombinant osteoprotegerin in the medium decreased the number of apoptotic (NF-B inactive) nuclei of RAECN2 plated on osteopontin in serum-free medium (squares). The control is endothelial cells plated on osteopontin with active NF-B (diamonds). Five random fields were counted per treatment, and the percentage of apoptotic nuclei was calculated for each field. The percentage of apoptotic nuclei was plotted against the concentration of osteoprotegerin. The error bars reflect S.D. values (n = 5).

Osteoprotegerin is a member of the TNF receptor superfamily. Osteoprotegerin contains a cysteine-rich amino-terminal domain, a putative death domain, and a COOH-terminal heparin-binding domain, but unlike other members of the TNF receptor family, does not contain a transmembrane domain. Therefore, it is thought to act as a soluble receptor (17, 20). Osteoprotegerin has been detected in bone, heart, lung, liver, stomach, placenta, calvaria, B cells, dendritic cells, and blood vessels (15, 17). Studies in mutant mice have validated the idea that osteoprotegerin is identical to the osteoblast-derived osteoclast inhibitory factor. Transgenic mice overexpressing osteoprotegerin exhibit increased bone density and increased mineralization due to a decrease in osteoclasts terminal differentiation (17). Osteoprotegerin null mice exhibit severe osteoporosis (15, 17, 21). Further studies have shown that osteoprotegerin inhibits osteoclastogenesis by binding osteoclast differentiation factor (RANKL/OPGL/TRANCE) and blocking its interaction with its receptor, RANK, on osteoclasts (TRANCE-R) (20, 22). In addition, a role for osteoprotegerin in development of germinal centers in secondary lymphoid tissues has been postulated (15).

Most relevant to the present report, osteoprotegerin has also been implicated as a cell survival factor. Osteoprotegerin was found to interact with TNF-related apoptosis-inducing ligand (TRAIL) (18). Osteoprotegerin was shown to bind to TRAIL with high affinity, and inhibited TRAIL-induced apoptosis of Jurkat cells (18). Our studies further support a role for osteoprotegerin in cell survival. We have shown that osteoprotegerin protects endothelial cells from apoptosis induced by serum withdrawal and NF-B inactivation. While the mechanism whereby osteoprotegerin protects endothelial cells is not yet clear, it is tempting to speculate that the effect of osteoprotegerin may be at the level of blocking a TRAIL or TRAIL-like death factor released under conditions of serum withdrawal.

Our studies are the first to identify the expression and survival function of osteoprotegerin in endothelial cells. The fact that osteoprotegerin induction is dependent on _v3 ligation suggests that osteoprotegerin may be involved in endothelial cell morphogenesis. Recently, _v3 has been implicated in angiogenesis, and blockade of _v3 blocks capillary formation by inducing endothelial cell death (1). Alternatively, regulation by NF-B suggests that osteoprotegerin may be involved in inflammatory functions of endothelial cells, since NF-B activation by various stimuli leads to up-regulation of inflammatory mediators and leukocyte adhesion molecules. These possibilities are underscored by the phenotype of the osteoprotegerin null mice that, in addition to severe osteoporosis, showed extensive inflammation and calcification of aorta and renal artery (23). Combined with our findings, these studies implicate osteoprotegerin as a potential regulator of vascular homeostasis, in addition to bone remodeling.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL18645 and DK47659 and National Science Foundation Grant EEC9529161.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: University of Washington, Box 351720, Seattle, WA 98195-1720. Tel.: 206-543-0205; Fax: 206-616-9763; E-mail: ceci@u.washington.edu.

Published, JBC Papers in Press, May 12, 2000, DOI 10.1074/jbc.C000290200

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; RAEC, rat aortic endothelial cell; PCR, polymerase chain reaction; HRP, horseradish peroxidase; FACS, fluorescence-activated cell sorter; PDL, poly-D-lysine; TRAIL, TNF-related apoptosis-inducing ligand.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. E. Kearns, S. Khosla, and P. J. Kostenuik Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease Endocr. Rev., April 1, 2008; 29(2): 155 - 192. [Abstract] [Full Text] [PDF]

				S. Efklidou, R. Bailey, N. Field, M. Noursadeghi, and M. K. Collins vFLIP from KSHV inhibits anoikis of primary endothelial cells J. Cell Sci., February 15, 2008; 121(4): 450 - 457. [Abstract] [Full Text] [PDF]

				G. Chidlow, J. P. M. Wood, J. Manavis, N. N. Osborne, and R. J. Casson Expression of Osteopontin in the Rat Retina: Effects of Excitotoxic and Ischemic Injuries Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 762 - 771. [Abstract] [Full Text] [PDF]

				P. Secchiero and G. Zauli The Puzzling Role of TRAIL in Endothelial Cell Biology Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): e4 - e4. [Full Text] [PDF]

				P. Secchiero, F. Corallini, E. Rimondi, C. Chiaruttini, M. G. di Iasio, A. Rustighi, G. Del Sal, and G. Zauli Activation of the p53 pathway down-regulates the osteoprotegerin expression and release by vascular endothelial cells Blood, February 1, 2008; 111(3): 1287 - 1294. [Abstract] [Full Text] [PDF]

				C.A. Allen, B.L. Hart, C.L. Taylor, and C.L. Clericuzio Bilateral Cavernous Internal Carotid Aneurysms in a Child with Juvenile Paget Disease and Osteoprotegerin Deficiency AJNR Am. J. Neuroradiol., January 1, 2008; 29(1): 7 - 8. [Full Text] [PDF]

				S. H. Mangan, A. V. Campenhout, C. Rush, and J. Golledge Osteoprotegerin upregulates endothelial cell adhesion molecule response to tumor necrosis factor-{alpha} associated with induction of angiopoietin-2 Cardiovasc Res, December 1, 2007; 76(3): 494 - 505. [Abstract] [Full Text] [PDF]

				M. Atteritano, H. Marini, L. Minutoli, F. Polito, A. Bitto, D. Altavilla, S. Mazzaferro, R. D'Anna, M. L. Cannata, A. Gaudio, et al. Effects of the Phytoestrogen Genistein on Some Predictors of Cardiovascular Risk in Osteopenic, Postmenopausal Women: A Two-Year Randomized, Double-Blind, Placebo-Controlled Study J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3068 - 3075. [Abstract] [Full Text] [PDF]

				G. Zauli, F. Corallini, F. Bossi, F. Fischetti, P. Durigutto, C. Celeghini, F. Tedesco, and P. Secchiero Osteoprotegerin increases leukocyte adhesion to endothelial cells both in vitro and in vivo Blood, July 15, 2007; 110(2): 536 - 543. [Abstract] [Full Text] [PDF]

				L. R. Rodrigues, J. A. Teixeira, F. L. Schmitt, M. Paulsson, and H. Lindmark-Mansson The Role of Osteopontin in Tumor Progression and Metastasis in Breast Cancer Cancer Epidemiol. Biomarkers Prev., June 1, 2007; 16(6): 1087 - 1097. [Abstract] [Full Text] [PDF]

				B. J. Bennett, M. Scatena, E. A. Kirk, M. Rattazzi, R. M. Varon, M. Averill, S. M. Schwartz, C. M. Giachelli, and M. E. Rosenfeld Osteoprotegerin Inactivation Accelerates Advanced Atherosclerotic Lesion Progression and Calcification in Older ApoE-/- Mice Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2117 - 2124. [Abstract] [Full Text] [PDF]

				J. Y. Shin, Y. G. Shin, and C. H. Chung Elevated Serum Osteoprotegerin Levels Are Associated With Vascular Endothelial Dysfunction in Type 2 Diabetes Diabetes Care, July 1, 2006; 29(7): 1664 - 1666. [Full Text] [PDF]

				G.-d. Xiang, L. Xu, L.-s. Zhao, L. Yue, and J. Hou The relationship between plasma osteoprotegerin and endothelium-dependent arterial dilation in type 2 diabetes. Diabetes, July 1, 2006; 55(7): 2126 - 2131. [Abstract] [Full Text] [PDF]

				W. J. Sandberg, A. Yndestad, E. Oie, C. Smith, T. Ueland, O. Ovchinnikova, A.-K. L. Robertson, F. Muller, A. G. Semb, H. Scholz, et al. Enhanced T-Cell Expression of RANK Ligand in Acute Coronary Syndrome: Possible Role in Plaque Destabilization Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 857 - 863. [Abstract] [Full Text] [PDF]

				M. Toruner, M. Fernandez-Zapico, J. J. Sha, L. Pham, R. Urrutia, and L. J. Egan Antianoikis Effect of Nuclear Factor-{kappa}B through Up-regulated Expression of Osteoprotegerin, BCL-2, and IAP-1 J. Biol. Chem., March 31, 2006; 281(13): 8686 - 8696. [Abstract] [Full Text] [PDF]

				L. M. Rasmussen, L. Tarnow, T. K. Hansen, H.-H. Parving, and A. Flyvbjerg Plasma osteoprotegerin levels are associated with glycaemic status, systolic blood pressure, kidney function and cardiovascular morbidity in type 1 diabetic patients Eur. J. Endocrinol., January 1, 2006; 154(1): 75 - 81. [Abstract] [Full Text] [PDF]

				X. Guang-da, S. Hui-ling, C. Zhi-song, and Z. Lin-shuang Changes in Plasma Concentrations of Osteoprotegerin before and after Levothyroxine Replacement Therapy in Hypothyroid Patients J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5765 - 5768. [Abstract] [Full Text] [PDF]

				C. S. Moran, M. McCann, M. Karan, P. Norman, N. Ketheesan, and J. Golledge Association of Osteoprotegerin With Human Abdominal Aortic Aneurysm Progression Circulation, June 14, 2005; 111(23): 3119 - 3125. [Abstract] [Full Text] [PDF]

				B. A. Mosheimer, N. C. Kaneider, C. Feistritzer, A. M. Djanani, D. H. Sturn, J. R. Patsch, and C. J. Wiedermann Syndecan-1 Is Involved in Osteoprotegerin-Induced Chemotaxis in Human Peripheral Blood Monocytes J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2964 - 2971. [Abstract] [Full Text] [PDF]

				C. A. Simmons, G. R. Grant, E. Manduchi, and P. F. Davies Spatial Heterogeneity of Endothelial Phenotypes Correlates With Side-Specific Vulnerability to Calcification in Normal Porcine Aortic Valves Circ. Res., April 15, 2005; 96(7): 792 - 799. [Abstract] [Full Text] [PDF]

				D. L. Courter, L. Lomas, M. Scatena, and C. M. Giachelli Src Kinase Activity Is Required for Integrin {alpha}V{beta}3-Mediated Activation of Nuclear Factor-{kappa}B J. Biol. Chem., April 1, 2005; 280(13): 12145 - 12151. [Abstract] [Full Text] [PDF]

				S. Patel, A. D. Leal, and D. H. Gorski The Homeobox Gene Gax Inhibits Angiogenesis through Inhibition of Nuclear Factor-{kappa}B-Dependent Endothelial Cell Gene Expression Cancer Res., February 15, 2005; 65(4): 1414 - 1424. [Abstract] [Full Text] [PDF]

				P. Collin-Osdoby Regulation of Vascular Calcification by Osteoclast Regulatory Factors RANKL and Osteoprotegerin Circ. Res., November 26, 2004; 95(11): 1046 - 1057. [Abstract] [Full Text] [PDF]

				T. Ueland, R. Jemtland, K. Godang, J. Kjekshus, A. Hognestad, T. Omland, I. B. Squire, L. Gullestad, J. Bollerslev, K. Dickstein, et al. Prognostic value of osteoprotegerin in heart failure after acute myocardial infarction J. Am. Coll. Cardiol., November 16, 2004; 44(10): 1970 - 1976. [Abstract] [Full Text] [PDF]

				A. Sahai, P. Malladi, X. Pan, R. Paul, H. Melin-Aldana, R. M. Green, and P. F. Whitington Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1035 - G1043. [Abstract] [Full Text] [PDF]

				K. Vidal, P. Serrant, B. Schlosser, P. van den Broek, F. Lorget, and A. Donnet-Hughes Osteoprotegerin production by human intestinal epithelial cells: a potential regulator of mucosal immune responses Am J Physiol Gastrointest Liver Physiol, October 1, 2004; 287(4): G836 - G844. [Abstract] [Full Text] [PDF]

M. Harada, Y. Osuga, T. Hirata, Y. Hirota, K. Koga, O. Yoshino, C.