Angiopoietin-like-4 is a potential angiogenic mediator in arthritis

Clinical Immunology 115 (2005) 93 – 101 www.elsevier.com/locate/yclim

Angiopoietin-like-4 is a potential angiogenic mediator in arthritis L.M. Hermanna, M. Pinkertona, K. Jenningsa, L. Yanga, A. Groma, D. Sowdersa, S. Kerstenb, D.P. Wittec, R. Hirschd, S. Thorntona,* b

a William S. Rowe Division of Rheumatology, Cincinnati Children’s Hospital, 3333 Burnet Avenue, ML 4010, Cincinnati, OH 45229, USA Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6700 EV, Wageningen, The Netherlands c Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA d Division of Rheumatology, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA

Received 23 September 2004; accepted with revision 7 December 2004 Available online 19 January 2005

Abstract Our previous studies of gene expression profiling during collagen-induced arthritis (CIA) indicated that the putative angiogenic factor Angptl4 was one of the most highly expressed mRNAs early in disease. To investigate the potential involvement of Angptl4 in CIA pathogenesis, Angptl4 protein levels were assessed at early stages of disease and its cellular sources were determined. In addition, the functional effects of mouse Angptl4 on endothelial cells were assessed. Angptl4 protein levels were higher in arthritic joints as compared to normal joints. In situ hybridization localized Angptl4 mRNA to stromal fibroblast-like cells within the inflamed synovium. Temporal expression of Angptl4 mRNA during CIA was similar to that of key angiogenic factors, including structurally related angiopoietin 1. Recombinant mouse Angptl4 promoted endothelial cell survival and formation of tubule-like structures. These functional effects of Angptl4, combined with very high expression at early stages of CIA, suggest a role for Angptl4 in angiogenesis in arthritis. D 2004 Elsevier Inc. All rights reserved. Keywords: Angptl4; Angiogenesis; Arthritis; Collagen-induced arthritis; Fibroblasts; Autoimmunity; Inflammation; Endothelial cells

Introduction Our previous microarray analysis identified Angptl4 as one of the most highly expressed genes early in collageninduced arthritis (CIA), a widely used model of rheumatoid arthritis (RA) [1]. The gene for Angptl4 has recently been identified by several groups and is also known as Fasting-Induced Adipose Factor (FIAF), PPARg Angiopoietin-Related Protein (PGAR), and Hepatic Fibrinogen/Angiopoietin-Related Protein (HFARP) [2–4]. Although an exclusive function for Angptl4 has not been assigned, limited data to date suggest that human Angptl4 may be involved in angiogenesis. These previous studies showed that human Angptl4 has antiapoptotic activity in vascular endothelial cells [3,5] and

* Corresponding author. Fax: +1 513 636 3328. E-mail address: [email protected] (S. Thornton). 1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2004.12.002

that human Angptl4 induces a pro-angiogenic response in the chicken chorioallantoic membrane assay [6]. Since angiogenesis is thought to play a major role in rheumatoid arthritis, the potential angiogenic activity of Angptl4 may be important in this disease. Within inflammatory synovial tissue, the formation of new blood vessels, or angiogenesis, provides nutrients to the growing inflammatory tissue, allows inflammatory cell infiltration, and produces chemokines via endothelial cells lining the vasculature [7,8]. The integral role of angiogenesis in the pathogenesis of arthritis is demonstrated by both the inhibition of arthritis via anti-angiogenic agents in animal models and the expression of key mediators of blood vessel formation in arthritic tissues. Administration of the angiogenesis inhibitor, AGM1470, suppresses established CIA in rats [9,10], and blockage of vascular endothelial growth factor (VEGF), one of the major inducers of blood vessel formation, inhibits arthritis in the CIA mouse

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model [11–15]. Gene transfer of angiostatin or endostatin, whose gene products are anti-angiogenic, or administration of K1–5, an angiostatin-like related inhibitor of angiogenesis, also has suppressive effects on inflammatory arthritis [16–18]. Blockage of Tie2, a receptor for Angiopoietin 1 and 2 (Ang1 and Ang2), in a CIA synovial window model also demonstrates inhibitory effects on angiogenesis occurring within arthritic tissue [19]. Several key angiogenic mediators are increased in arthritis. Increased levels of vascular endothelial growth factor (VEGF) are observed in RA synovial fluid and tissues [20], CIA joint tissues [14,15], and juvenile rheumatoid arthritis (JRA) synovial tissues [21]. Angiopoietins are also major players in the stabilization and formation of new blood vessels [22]. Expressions of the angiopoietins, Ang1 and Ang2, and the angiopoietin receptors, Tie1 and Tie2, are increased in RA tissues as compared to osteoarthritis (OA) synovial tissues [23]. Gravallese et al. have also detected stronger Ang1 expression by immunohistochemistry in synovial tissues of RA patients than in OA tissues [24]. Thus, the more inflammatory RA disease seems to have higher expression of key angiogenic molecules than that observed in OA synovial tissues [21,23,24], suggesting that the overall angiogenic activity appears to parallel the extent of inflammation. Angptl4 is a secreted protein that is structurally similar to members of the angiopoietin family, which all contain N-terminal coiled-coil domains and C-terminal fibrinogenlike domains; however, Angptl4 does not bind either the Tie1 or Tie2 angiopoietin receptors [3]. Mouse Angptl4 shares 75% nucleotide identity and 77% amino acid identity with human Angptl4 [3], suggesting that Angptl4 may function similarly in both species. Similar to the anti-apoptotic effects of Ang1, human recombinant Angptl4 is able to reduce apoptosis of human umbilical vein endothelial cells (HUVEC) in culture, but not other cell types, including, cardiac fibroblasts, vascular smooth muscle cells, renal mesangial cells, HeLa cells, and HepG2 cells [3]. Thus, Angptl4’s anti-apoptotic effects appear to be specific for endothelial cells. In addition, human Angptl4 may also promote blood vessel formation as it is able to stimulate the formation of tubules from endothelial cells [5]. Interestingly, the tissue distribution of Angptl4 expression appears to be more limited than that of Ang1. Whereas Ang-1 is expressed ubiquitously in adult tissues [25,26], Angptl4 mRNA expression has been shown to be limited to specific tissues, including liver, kidney, adipose tissue [2,3], and as reported here, inflamed synovium. Furthermore, very little is known about the specific cells that express Angptl4. To analyze the role of mouse Angptl4 in arthritic processes, we have determined the expression of Angptl4 mRNA and protein levels at different stages of CIA and compared these to the mRNA levels of several key angiogenic mediators, including VEGF and Ang1. Our

analysis also localized Angptl4 transcripts to stromal fibroblast-like cells in mouse arthritic tissues. Furthermore, we have expressed a recombinant form of mouse Angptl4 and analyzed its potential function in angiogenesis by assessing its ability to stimulate tubule formation of endothelial cells in vitro. Taken together, our results suggest that consistent with the data on human Angptl4 [3,5,6], mouse Angptl4 has functional effects on endothelial cells and, thus, is likely to have a role in both angiogenesis and arthritis. In addition, these studies support the use of the CIA mouse model for the analysis of angiogenesis in arthritis.

Materials and methods Mice Male DBA/1J mice, 6–8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in the animal care facility at Cincinnati Children’s Hospital Research Foundation (Cincinnati, OH) under the Institutional Animal Care and Use Committee approved conditions. Collagen immunization Mice were injected intradermally with 100 Ag of bovine collagen type II (CII) (Elastin Products Co., Inc., Owensville, MO) in complete Freund’s adjuvant (CFA) at the base of the tail on day 0 and a similar booster was administered on day 21. Mice were evaluated for arthritis using an established macroscopic scoring system ranging from 0 to 4 (0 = no detectable arthritis, 1 = swelling and/ or redness of paw or one digit, 2 = two joints involved, 3 = three or four joints involved, and 4 = severe arthritis of the entire paw and digits) [27–30]. Mice were sacrificed at specified time points following primary collagen immunization, after which paws with an arthritic score of four were removed for mRNA analysis and in situ hybridizations (ISH). Paws from mice of the same age but not treated with CII were used as normal controls. DNA microarray analysis Microarray analysis has previously been described in detail [1]. Briefly, mRNA of a whole 1-day-old mouse was used for normalization of gene expression levels across all microarray chips. Competitive hybridizations with Cy3labeled whole 1-day-old mouse mRNA versus Cy5-labeled paw mRNA (pooled from 3–4 mouse paws/sample) were performed. Each Cy5-labeled normal day 28 or day 49 paw mRNA was hybridized together with the Cy-3-labeled whole 1-day mouse mRNA to two microarray chips for technical replication. Hybridizations were performed on the mouse GEM1 array by Incyte Genomics (Palo Alto, CA). Primary

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data were examined using Incyte Gemtools software and GeneSpring version 4.0.4 software (Silicon Genetics, Redwood City, CA).

IgG-HRP (Southern Biotechnology Associates), diluted 1:1000 in blocking buffer, developed, and detected as above.

In situ hybridization

RNase protection assays (RPA)

Mouse tissues were fixed for 48 h in 4% (w/v) paraformaldehyde in PBS at 48C immediately after harvesting. Following fixation, tissue was decalcified in TBD-2 (ThermoShandon, Pittsburgh, PA), with complete decalcification being determined by the use of 5% ammonium oxalate. Following decalcification, the tissue was rinsed for 10 min in running water and placed in 30% sucrose in PBS for 24 h at 48C. Samples were embedded in M-1 mounting media (Shandon), frozen in liquid nitrogen, and stored at 808C. Ten-micron cryostat sections of frozen tissue were air dried on TESPA-coated Superfrost Plus (Histology Control Systems, Glenhead, New York) slides and postfixed in 4% (w/v) paraformaldehyde in PBS then acetylated with acetic anhydride. Mouse anti-sense and sense Angptl4 probe templates were generated by linearizing the Incyte clone, GenBank W13905. Probes were generated by in vitro transcription. Slides were hybridized overnight at 458C under a sealed coverslip. Following hybridization, the sections were treated with RNase to remove unbound probe and slides were washed extensively. Slides were developed, counterstained with hematoxylin and eosin, and photographed using both dark- and bright-field illumination.

Quantitation of mouse Ang1, VEGF, CD31, TIE1, TIE2, and Flt1 mRNA levels was performed on 5 Ag of total paw RNA using the mouse Angio-1 multi-probe template (BD Biosciences Pharmingen, San Diego, CA). (a-32P) UTP-labeled anti-sense RNA probes were synthesized by in vitro transcription of these cDNA templates using BD Riboquant In Vitro Transcription Kit (BD Biosciences Pharmingen). DNA templates were degraded by DNase I digestion and probes were purified by phenol/chloroform extraction and ethanol precipitation with subsequent hybridization to total RNA at 568C overnight. Samples were treated with RNase A + T1 and double-stranded RNA was purified by phenol/chloroform extraction and precipitated with ethanol and salt. Samples were resuspended in loading dye and electrophoresed on a 5% denaturing polyacrylamide gel. The gel was dried and subjected to Phosphorimager analysis using a Storm 860 and Imagequant software (Molecular Dynamics, Sunnyvale, CA). The mRNA levels are expressed as the ratio of the Phosphorimager units of the specified gene to those of GAPDH from the same RNA sample multiplied by 100 (100).

Western analysis

Protein purification and Matrigel tubule formation assays

Paws were quick frozen and stored at 708C until use. Tissue was placed in 1 ml lysis buffer (PBS, 1% Triton X-100, 2 mM PMSF) and homogenized with a PowerGen 700 homogenizer (Fisher Scientific, Pittsburgh, PA). Cellular debris were pelleted and supernatant containing protein was stored in aliquots at 708C. Two hundred micrograms of tissue extract was separated by SDS–PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked overnight in 5% milk, 0.1% Tween 20, TBS, and incubated with antiAngptl4 antibody diluted 1:1000 in blocking buffer. The membrane was washed and subsequently incubated with anti-rabbit IgG-HRP (Sigma, St Louis, MO) diluted 1:8000 in blocking buffer, developed with chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ), and exposed to X-ray film for detection. For detection of recombinant mouse Angptl4 (rmAngptl4), anti-His Ab (Tetra-His, Qiagen, Valencia, CA) was used at a concentration of 1:1000, with secondary antibody (goat anti-mouse IgG-HRP, Southern Biotechnology Associates, Birmingham, AL) used at a concentration of 1:4000. Anti-tubulin antibody was kindly provided by Dr. James Lessard, Cincinnati Children’s Hospital, Cincinnati, OH, and was used at a concentration of 1:1000 diluted in blocking buffer, subsequently probed with anti-mouse

The cDNA encoding mouse Angptl4 was placed into the expression vector pcDNA3.1mycHisA generating the construct pcDNA3.1mycHisrmAngptl4. HEK293 cells were stably transfected with this plasmid and supernatant was collected from cells grown in DMEM supplemented with 2% FCS, 50 U/ml penicillin, 50 Ag/ml streptomycin, and 1 mg/ml geneticin. Recombinant mouse Angptl4 (rmAngptl4) was purified as previously described by Kim et al. [3]. Briefly, supernatant from stably transfected cells or nontransfected cells was concentrated by centrifugation with Centricon YM-30 (Millipore Corporation, Bedford, MA) and incubated with Ni-nitroacetate agarose beads (Qiagen). Beads were washed with 10 mM imidazole and rmAngptl4 was eluted with 250 mM imidazole. Following elution, protein was concentrated by centrifugation with Centricon YM-30 (Millipore Corporation) and buffer exchange into 1 PBS. Growth factor-reduced Matrigel (BD Pharmingen) was plated on 24-well plates (300 Al/well). After 3 h of solidification at 378C, HUVECs were plated in EBM-2, 2% FCS without growth factors (Cambrex Bio Science Walkersville, Inc., Walkersville, MD), and supplemented with one of the following: 125 ng human fibroblast growth factor (hFGF); PBS; purified rmAngptl4, or purified protein from untransfected 293 cells.

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Results Angptl4 mRNA and protein levels are increased early in CIA The onset of CIA occurs approximately 26–28 days following primary collagen immunization. Angiogenic events likely occur at early stages of CIA, as both VEGF and von Willebrand factors were previously shown to be expressed early in disease [15]. Microarray analysis detected Angptl4 mRNA in normal and arthritic paws. Angptl4 mRNA levels were increased approximately 10-fold in arthritic paws at day 28 of disease (Fig. 1) as compared to normal joints with Angptl4 mRNA levels decreasing late in disease. To determine whether Angptl4 protein levels were similar to the observed increase in Angptl4 mRNA levels early in CIA, Western analysis using an antibody specific to Angptl4 was performed on mouse whole paw extracts (Fig. 2). Since Angptl4 has been detected previously in liver [2], extracts from liver were used as a positive control for Western analysis. Arthritic day 28 paw extracts exhibited much higher levels of Angptl4 than those from normal mouse joints, demonstrating the increased expression of Angptl4 protein early in CIA. The migration of Angptl4 protein in paw extracts is very similar to that of liver; however, a slight difference in molecular weight may exist which could reflect glycosylation or other post-translational modifications, which have been observed with human and rat Angptl4 [3,31].

mouse tissues were subjected to in situ hybridization. Angptl4 mRNA was detected in stromal, spindle-shaped, fibroblast cells within mouse paws at day 28 of disease (Figs. 3A–B and D–E). The signal was restricted to fibroblasts in the proliferating granulation tissue. No signal was detected in the endothelium lining the blood vessels (Figs. 3A and B) or in the infiltrating neutrophils. Surrounding skeletal muscle fibers, bone, and quiescent fibroblasts in the tendons and joint capsule were also negative. Although we have detected low levels of Angptl4 protein and mRNA in normal paws by other methods, no signal for Angptl4 transcripts was detectable in sections of normal paws (Fig. 3F). In addition, no signal was detected in day 28 paw sections hybridized with the sense Angptl4 RNA probe (Fig. 3C).

Angptl4 message is localized to stromal, fibroblast cells in CIA joints

Key angiogenic mediators are expressed early in CIA similarly to Angptl4

In our initial experiments, Angptl4 mRNA was detected using total RNA extracted from whole synovial tissues. Further characterization of the cellular source of Angptl4 mRNA would both confirm our microarray mRNA results and provide information about the potential role of Angptl4 in disease. To localize Angptl4 transcripts, CIA and normal

Since human Angptl4 has been proposed to have angiogenic activity in vitro and in vivo, we analyzed the expression of other key angiogenic mediators during CIA by RNase protection analysis. As shown in Fig. 4, mRNA levels of several molecules associated with angiogenesis including Ang1, VEGF, CD31, TIE2, TIE1, and Flt1 are increased early in CIA as compared to normal paws and decrease later in disease. Thus, these key angiogenic mediators exhibit temporal expression patterns similar to Angptl4 expression observed in arthritic joints.

Fig. 2. Angptl4 protein expression in normal and arthritic mouse paws. Western analysis on paw extracts (200 Ag) from either normal mice or paws with a score of 4 from day 28 following primary CII immunization (4 animals each) was performed with either anti-Angptl4 antibody or antiTubulin antibody as indicated. Liver extracts (300 Ag) serve as positive control for Angptl4 protein expression. Tubulin is used as a loading control.

Recombinant mouse Angptl4 (rmAngptl4) induces tubule formation in human umbilical vein endothelial cells (HUVEC)

Fig. 1. Angptl4 mRNA levels are increased early in CIA. Normal nonimmunized DBA/1 mice and mice at day 28 or 49 following primary CII immunization were sacrificed and paws were taken for RNA isolation. mRNA was pooled from 4 paws of normal or arthritic mice, labeled, and hybridized to two identical Incyte Gem1 microarray chips. Bars represent values for Angptl4 mRNA levels for individual chips. The value for Normal (1) is set at 1 and all other values are represented as the fold change as compared to this sample.

Human Angptl4 has been shown to induce tubule formation from endothelial cells. To determine whether mouse Angptl4 has angiogenic properties, we assessed its ability to induce tubule formation in endothelial cells. To generate rmAngptl4, the cDNA encoding mouse Angptl4 was cloned into the mammalian expression vector, pcDNA3.1mycHisA (Invitrogen, Carlsbad, CA) (Fig. 5). Western analysis of the purified protein from supernatant of HEK293 cells stably transfected with pcDNA3.1mycHisr-

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Fig. 3. Localization of Angptl4 expression in arthritic tissues. Mouse day 28 of CIA (A–E); normal mouse (F). Panels A and B show the expression of Angptl4 (bright white grains in Panel A) in the granulation tissue surrounding the bone (bo). No signal was detected in the bone, blood vessels (*), or muscle cells (mu). Panels D and E show expression in the periosteal tissue (arrows). Panel C shows day 28 of CIA hybridized with the sense control probe and panel F shows normal mouse tissue hybridized with the antisense probe, both of which show no detectable signal. The * in panel F indicates the joint lined by normal synovial tissue. (Magnification: Panels A–B and D–E, 200, and Panels C and F, 100; Illumination: Panels A, C, D, and F, dark field, and Panels B and E, bright field.

mAngptl4 indicates the presence of the approximately 56 kDa rmAngptl4 as assessed with either anti-His or antiAngptl4 antibody (Fig. 5). A smaller molecular weight fragment is also observed with the anti-His antibody, which may be generated through proteolytical processing, as has been observed for recombinant rat Angptl4 [31]. Purified rmAngptl4 was subsequently tested for its ability to induce tubule formation of HUVEC. As shown in Fig. 6, rmAngptl4 induces the formation of tubule structures in HUVEC cultured on growth factor-reduced Matrigel in a concentration-dependent manner; whereas, protein purified from non-transfected 293 cell supernatant does not induce tubule formation. These results suggest a functional role for mouse Angptl4 in vascular formation and/or stabilization.

Discussion Based on our earlier study of gene expression profiling of inflamed joints over the course of CIA, Angptl4 was one of the most highly increased mRNAs early in disease. The

present study confirmed the increased expression of Angptl4 at both the mRNA and protein levels at early stages of CIA. This observed increase in Angptl4 mRNA expression was similar to that of other key angiogenic mediators, including VEGF and Ang1. Angptl4 mRNA expression was also localized to stromal, fibroblast cells in mouse arthritic tissues. Furthermore, the angiogenic potential of mouse Angptl4 was demonstrated through its induction of tubule formation in HUVEC. Although increased Angptl4 expression has been demonstrated in certain tumors, to our knowledge, the present study is the first demonstration of increased expression of Angptl4 in inflammatory conditions. This observation suggests that Angptl4 may have a role in the pathogenesis of arthritis. Since Angptl4 is highly similar to the angiopoietins structurally, it is not surprising that human Angptl4, like Ang1, has anti-apoptotic effects on endothelial cells [3], which can lead to stabilization of newly formed blood vessels. Consistent with this, human Angptl4 has been shown to promote survival of endothelial cells and blood vessel formation in in vivo experimental systems [5].

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Fig. 4. mRNA levels of specific angiogenic molecules in normal and arthritic mouse paws. Total RNA isolated from whole paws of either normal mice or mice at day 28 and day 49 of CIA (paws with a score of 4) was assessed by RNase protection analysis for the expression levels of the indicated molecules and GAPDH. Values are the indicated mRNA phosphorimager values divided by the GAPDH values for that sample multiplied by 100. Bars represent the mean values F the SEM of 4 separate paw RNAs.

The formation and stabilization of new blood vessels within inflammatory tissues is important to the pathogenesis of arthritis. Indeed, new blood vessels can provide nutrients to the growing tissue. In addition, endothelial cells lining these blood vessels can secrete chemokines to recruit inflammatory cells into inflamed tissues and also allow migration of inflammatory cells into the inflamed tissue. Our observations that rmAngptl4 can induce HUVEC to form tubules in the absence of other endothelial cell growth factors demonstrate that mouse Angptl4 has specific effects on endothelial cells and further support its role in angiogenesis in a setting of inflamed arthritic tissue. Within inflamed synovium, in situ hybridization localized Angptl4 mRNA expression to stromal, fibroblast cells adjacent to blood vessels. Angptl4 is a secreted protein that is likely to have paracrine effects on endothelial cells. Therefore, expression and secretion of Angptl4 from stromal, fibroblast cells in the synovium could provide angiogenic signals to endothelial cells and thus promote or stabilize blood vessel formation within the expanding inflammatory synovial tissue. The intense signal given by stromal fibroblast cells in arthritic paws indicates that Angptl4 mRNA is produced in large amounts by these cells in arthritic, inflamed joints. In contrast, the expression of Angptl4 in normal mouse paws appears to be minimal. In the present study, Angptl4 was detected at low levels by Western and microarray analysis, where whole paws were used. By in situ hybridization analysis, which used only sections of mouse paws, Angptl4 was not detected at all in normal paws, suggesting that Angptl4 expression is a feature of inflammatory processes. Our data showed that Angptl4 expression paralleled the temporal expression patterns of key angiogenic molecules at early stages of CIA. Angptl4, Ang1, VEGF, CD31, TIE1, TIE2, and Flt1 were all expressed at a higher level early in disease than in normal mouse paws, and levels of these

mRNAs decreased at day 49 of CIA. This is consistent with the observed increase in inflammatory cytokines early in disease [27] and the expression of VEGF and von Willebrand factor (a widely-used marker of vascularity) observed by Lu et al. early in disease [15]. Furthermore, not only is Angptl4 expressed similar to other angiogenic molecules, but also its tissue distribution is limited to only a few tissues, including inflamed synovium, liver, kidney, and adipose tissue. Thus, the similar temporal expression of Angptl4 with other angiogenic molecules and its limited tissue expression as compared to other angiogenic molecules suggest that Angptl4 may play a specific role in the angiogenic processes that contribute to arthritis. Increased expression of Angptl4 has also been demonstrated in certain human tumors, another pathological phenomenon that depends on neovascularization [6]. This observation is interesting in that RA synovial fibroblasts have been characterized as having a transformed, btumorlikeQ phenotype [32]. Expression of Angptl4 signal appears to localize to these proliferating fibroblasts within CIA synovium, suggesting that the increased expression of Angptl4 may contribute to the transformed phenotype of synovial fibroblasts. Angiogenesis is an integral part of arthritic processes in both RA and CIA, with potentially several angiogenic factors contributing to disease progression. Recently, the expression of placental growth factor (PlGF) in ischemic tissues has been shown to contribute to pathogenic angiogenesis and potentially arthritic processes [33]. PlGF exerts its effects through the VEGF receptor Flt1, and administration of anti-Flt-1 agents has been shown to result in a decrease in angiogenesis and inflammatory joint destruction in experimental arthritis. Autiero et al. have also recently shown that Angptl4 mRNA levels are increased in response to PlGF treatment in endothelial cells, suggesting that

Fig. 5. Generation and purification of recombinant mouse Angptl4 (rmAngptl4). Panel A shows a schematic of rmAngptl4 expression construct. The entire cDNA for mouse Angptl4 is represented by the open box and the closed box at the carboxy terminus represents the myc/His tag. Panel B shows Western analysis of purified protein from supernatants of 293 cells either stably transfected with rmAngptl4 (lanes 3 and 5) or nontransfected 293 cells (lanes 2 and 4). A His-tagged ladder is present in lane 1. rmAngptl4 was detected with either an anti-His (lanes 1–3) antibody or an anti-Angptl4 (lanes 4–5) antibody.

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Fig. 6. Purified rmAngptl4 induces tubule formation of HUVEC cells. HUVECs were plated on growth factor-reduced Matrigel. Cells were grown in EBM-2 without growth factors containing 2% FCS with the following supplements: (A) 125 ng FGF; (E) 50 Al PBS; (B, C, and D) 10, 20, and 50 Al of purified protein from nontransfected 293 cell supernatant; (F, G, and H) 10, 20, and 50 Al of purified protein from rmAngptl4 stably transfected 293 cell supernatant. HUVECs were examined 24 h after plating and pictures were taken of the middle of the wells at 4 magnification. Figure is representative of duplicate wells of one of three experiments.

Angptl4 may be one of the downstream modulators of PlGF-regulated angiogenesis [34]. In summary, the present study demonstrates the increased expression of Angptl4 in synovial fibroblasts within arthritic tissue. To our knowledge, this is the first description associating expression of this angiogenic molecule with inflammatory processes. Our studies also indicate that the temporal expression of Angptl4 mRNA during CIA is

similar to other key angiogenic mediators, including Ang1 and VEGF, further supporting a role for Angptl4 in angiogenesis and arthritis. In addition, functional analysis of mouse Angptl4 demonstrates its ability to induce tubule formation of endothelial cells, similar to that of human Angptl4. Given its previously described limited tissue distribution, Angptl4 makes an attractive target for potential therapeutic inhibition of angiogenesis in arthritic tissue.

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Based on our data, the CIA model provides an appropriate setting for analysis of Angptl4 in angiogenesis and arthritis. Further studies will define more precisely mechanisms of the functional role of Angptl4 in arthritis.

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Acknowledgments We would like to thank Drs. David Glass and Michael Barnes for their helpful discussions of the manuscript and Lorie Luyrink for technical assistance. Supported in part by the Arthritis Foundation, NIH grants AR47784, AR47363, The Schmidlapp Foundation, and the Cincinnati Children’s Hospital Research Foundation.

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