Molecular Medicine Milk Fat Globule Protein Epidermal Growth Factor-8 A Pivotal Relay Element Within the Angiotensin II and Monocyte Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth Muscle Cells Invasion Zongming Fu,* Mingyi Wang,* Marjan Gucek, Jing Zhang, James Wu, Liqun Jiang, Robert E. Monticone, Benjamin Khazan, Richard Telljohann, Julie Mattison, Simon Sheng, Robert N. Cole, Gaia Spinetti, Gianfranco Pintus, Lijuan Liu, Frank D. Kolodgie, Renu Virmani, Harold Spurgeon, Donald K. Ingram, Allen D. Everett, Edward G. Lakatta,* Jennifer E. Van Eyk* Downloaded from http://ahajournals.org by on June 14, 2020 Abstract—Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis. (Circ Res. 2009;104:1337-1346.) Key Words: MFG-E8 䡲 angiotensin II 䡲 monocyte chemoattractant protein-1 䡲 vascular smooth muscle cells 䡲 aging P roinflammatory processes and associated elevated invasion capacity of vascular smooth muscle cells (VSMCs) are increased within the diffuse thickening of the arterial wall that evolves with advancing age.1– 4 In humans, this ageassociated arterial remodeling is an independent risk factor for the epidemic of quintessential human cardiovascular diseases, ie, atherosclerosis, hypertension, and stroke.1,3,5,6 The age-associated arterial wall thickening and other aspects of arterial remodeling are evolutionarily conserved in various mammalian species, including rodents, nonhuman primates, and humans.1,3,7–14 The thickened arterial intima is formed because of VSMCs invasion and proliferation and is not limited to secretion within the subendothelial space.2,3,7,14 A growing body of evidence indicates that VSMCs within the arterial media begin to express and activate proteases such as matrix metalloprotease (MMP)2 and calpain-1, which enable cytoskeletal remodeling and degradation of (1) basement membranes that surround VSMCs, (2) adjacent matrix, and (3) elastic lamina. Thus VSMCs invasion into the subendothelial space is driven, at least in part, by angiotensin (Ang) Original received September 8, 2008; revision received April 28, 2009; accepted May 4, 2009. From the Department of Medicine (Z.F., S.S., J.E.V.E.); Technical Implementation and Coordination Core (M.G., R.N.C., J.E.V.E.), The Johns Hopkins Bayview Proteomics Center; Department of Neurology (L.L.); Department of Pediatrics (A.D.E.); Department of Biomedical Engineering (J.E.V.E.); and Department of Biological Chemistry (J.E.V.E.), The Johns Hopkins University, Baltimore, Md; Laboratory of Cardiovascular Science (M.W., L.J., R.E.M., B.K., R.T., H.S., E.G.L.) and Laboratory of Experimental Gerontology (J.M.), National Institute on Aging, NIH, Baltimore, Md; Medstar Research Institute (J.Z., J.W.), Hyattsville, Md; IRCCS MultiMedica (G.S.), Milano, Italy; Department of Biomedical Sciences (G.P.), University of Sassari, Italy; CVPath (F.D.K., R.V.), International Registry of Pathology, Gaithersburg, Md; and Nutritional Neuroscience and Aging Laboratory (D.K.I.), Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge. This manuscript was sent to Joseph Loscalzo, Consulting Editor, for review by expert referees, editorial decision, and final disposition. *These authors contributed equally to this work. Correspondence to Mingyi Wang, MD, PhD, Laboratory of Cardiovascular Science, National Institute on Aging-National Institutes of Health, Baltimore, MD 21224. E-mail: mingyiw@grc.nia.nih.gov © 2009 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.187088 1337 1338 Circulation Research Table. June 19, 2009 Differentially Abundant Proteins Identified by 2-D DIGE and iTRAQ Protein Class and Protein Name Accession* iTRAQ† O/Y DIGE‡ O/Y PTM§ MFGM_RAT 2.31⫾0.21 2.53⫾0.15 Glycosylation Apoptosis/cell cycle/proliferation MFG-E8 Serine protease HTRA1 HTRA1_MOUSE 2.03⫾0.17 ND Integral membrane protein 2B ITM2B_RAT 2.28⫾0.63 ND Cysteine and glycine-rich protein 2 CSRP2_RAT 1.56⫾0.08 ND PLF4_RAT 3.09⫾1.5 ND IBP7_MOUSE 1.40⫾0.2 ND Platelet factor 4 (CXCL4) Insulin-like growth factor-binding protein 7 (IGFBP-7) Cell Metabolism Creatine kinase M-type KCRM_RAT 0.58⫾0.02 ND Glycerol 3-Pdehydrogenase 1 GPDA_RAT 0.58⫾0.10 0.52⫾0.08 Cytochrome c oxidase subunit 5B COX5B_RAT 0.66⫾0.02 ND Glutamine synthetase GLNA_RAT 0.58⫾0.10 ND 2-oxoglutarate dehydrogenase E1 component ODO1_RAT 0.67⫾0.11 ND 2,4-dienoyl-CoA reductase DECR_RAT 0.63⫾0.06 ND 3-ketoacyl-CoA thiolase THIM_RAT 0.66⫾0.07 0.60⫾0.07 Hydroxyacyl-coenzyme A dehydrogenase HCDH_RAT 0.61⫾0.12 ND Trifunctional enzyme subunit ␤ ECHA_RAT 0.66⫾0.07 ND Long-chain-fatty-acid–CoA ligase 1 ACSL1_RAT 0.63⫾0.12 0.62⫾0.07 Isocitrate dehydrogenase [NADP] IDHP_RAT 0.75⫾0.05 0.65⫾0.05 Pyruvate carboxylase PYC_RAT 0.93⫾0.02 0.56⫾0.07 RBP1_RAT 1.49⫾0.02 ND Actin-related protein 2/3 complex subunit 4 ARPC4_MOUSE 1.54⫾0.07 ND STE20-like serine/threonine-protein kinase SLK_RAT 0.59⫾0.03 ND Calponin-1 CNN1_RAT 1.42⫾0.06 1.69⫾0.06 Calponin-3 CNN3_RAT 1.45⫾0.07 1.75⫾0.06 Cytoskeleton/invasion/migration RalA-binding protein 1 Downloaded from http://ahajournals.org by on June 14, 2020 Phosphorylation Extracellular matrix/cell adhesion Transforming growth factor ␤-3 TGFB3_RAT 2.22⫾0.21 ND Clusterin CLUS_RAT 1.89⫾0.26 ND 72 kDa type IV collagenase (MMP-2) MMP2_RAT 1.40⫾0.11 ND Biglycan PGS1_RAT 1.56⫾0.06 ND Collagen ␣-1(I) chain¶ CO1A1_RAT 0.39⫾0.11 0.35⫾0.05 Collagen alpha-2(I) chain¶ CO1A2_RAT 0.54⫾0.14 0.45⫾0.06 FINC_RAT 1.68⫾0.10 2.1⫾0.19 ND Fibronectin 1 SPARC-like protein 1 SPRL1_RAT 1.47⫾0.14 VTNC_MOUSE 1.91⫾0.09 ND Apolipoprotein A-I APOA1_RAT 0.68⫾0.13 0.60⫾0.04 Kininogen 1 KNT1_RAT 1.67⫾0.22 1.86⫾0.19 Glycosylation Periostin (OSF-2) POSTN_MOUSE 2.43⫾0.13 2.46⫾0.19 Glycosylation Apolipoprotein E APOE _RAT 1.55⫾0.29 ND Fetuin-A; ␣-2-HS-glycoprotein FETUA_RAT 0.61⫾0.20 ND Fetuin-B FETUB_RAT 0.66⫾0.05 ND Vitronectin (Continued) *Accession is from SwissProt database; †changes in abundance with P⬍0.05 and ⬎1.5-fold in 4 animals in each group using iTRAQ; ‡changes in abundance with P⬍0.05 and ⬎1.5-fold in 8 animals in each group using 2DE (ND indicates not detected by this method); §potential PTM based on PNGase F and phosphostaining experiments (see the online data supplement); ¶soluble form of collagen decreased, whereas the insoluble form that is known to increase with aging will not be able to get observed by 2DE or iTRAQ. Fu et al Table. MFG-E8 Mediates VSMC Invasion 1339 Continued Protein Class and Protein Name Accession* iTRAQ† O/Y DIGE‡ O/Y Adenylate cyclase type 5 ADCY5_RAT 1.54⫾0.08 ND ␣-1-antiproteinase A1AT_RAT 0.66⫾0.09 0.58⫾0.09 ␣-1-inhibitor III A1I3_RAT 0.67⫾0.14 0.60⫾0.08 Carbonic anhydrase 3 CAH3_RAT 0.63⫾0.23 ND Carboxylesterase 1 EST2_RAT 0.49⫾0.03 ND Contrapsin-like protease inhibitor 1 SPA3K_RAT 0.66⫾0.05 ND Contrapsin-like protease inhibitor 3 SPA3L_RAT 0.62⫾0.10 ND Ferritin light chain FRIL1_RAT 1.56⫾0.12 1.91⫾0.09 Major urinary protein MUP_RAT 0.46⫾0.04 ND Solute carrier family 2; facilitated glucose transporter member 4 GTR4_RAT 2.01⫾0.02 ND Transthyretin TTHY_RAT 0.59⫾0.18 0.63⫾0.12 TRI47_MOUSE 1.97⫾0.09 ND PTM§ Miscellaneous Tripartite motif-containing protein 47 Downloaded from http://ahajournals.org by on June 14, 2020 II.7–14 VSMC invasion is also facilitated by an intimal–medial concentration gradient of monocyte chemoattractant protein (MCP)-1, and platelet-derived growth factor (PDGF)-BB.14 Transcription, translation, and activation of MMP2, calpain-1, MCP-1, and PDGF-BB are linked to an ageassociated increase in local arterial Ang II signaling via the Ang II type 1 receptor.7–14 However, numerous yet identified proteins that have crucial roles in the coordinated VSMCs invasion process likely change in abundance or become posttranslationally modified with aging. A complete understanding of mechanisms involved in the increased VSMCs invasive capacity with aging requires identification of the proteome changes within functional classes and characterization of interconnections between known pathways, as well as indicating previously unknown key regulators. To this end, we have performed a comprehensive quantitative proteomic study using both 2D gel electrophoresis (2DE)-based and mass spectrometry (MS)-based iTRAQ (isobaric tag for relative and absolute quantification) approaches to analyze aortic proteins obtained from young (8 months) and old (30 months) rats (schematic diagram of proteomic experimental design; Figure I in the online data supplement, available at http://circres.ahajournals. org). Furthermore, we used 2DE in combination with Pro-Q Diamond Phosphoprotein Gel Stain and deglycosylation experiments to characterize selected posttranslational modifications of proteins. Using these approaches in combination with quantitative RT-PCR, and Western blotting, immunostaining, and VSMCs functional assays, we have discovered (1) that at least 50 proteins change abundance in the aorta associated with aging; (2) that milk fat globule protein epidermal growth factor 8 (MFG-E8) (also known as lactadherin) markedly increases with aging, not only in rat aorta but also in nonhuman primate and human aorta; and (3) that MFG-E8 is a novel link between proinflammatory molecules Ang II and MCP-1 signaling and promotes the invasive capacity of VSMCs. Materials and Methods For details on the materials and methods used in the present study (including arterial specimens, isolation of aorta and preparation of Glycosylation aortic proteins, 2D silver-stained and DIGE, in-gel tryptic digestion and peptide desalting, mass spectrometry analysis and protein identification, detection of phosphoproteins and glycoproteins, iTRAQ analysis, Ingenuity pathway analysis, real-time PCR analysis, Western blotting analysis, immunohistochemistry and immunofluorescence, VSMCs isolation and culture, MFG-E8 small interfering [si]RNA silencing, VSMCs invasion assay, and statistical analysis), see the online data supplement, available at http://circres.ahajournals.org. Results Quantification and Identification of the Aortic Proteome by 2DE and iTRAQ MS-Based Analysis Using 2DE (silver-stained and DIGE gels), we have obtained 2D gel maps of 286 identified nonredundant proteins from rat aorta and observed 18 proteins (Table) that significantly change abundance (⫾1.5-fold) with aging (annotated protein gel maps for young and old aorta are shown in Online Figures II and III; corresponding to identifications listed in Online Table II). Using iTRAQ, 880 proteins were quantified and between both methods, 50 proteins were shown to have significantly different abundance associated with aging (⫾1.5-fold) (Online Table III). The results of the 2 methods were complimentary, increasing the confidence of the results. Totally 923 nonredundant proteins were detected by 2 methods. The majority of the proteins detected by 2DE (243 [85%]) are also quantified by iTRAQ. Approximately 5% (50) of the proteins identified have significantly different abundance with aging (Table). Of the proteins found to differ with aging, 5 had posttranslational modifications. Acidic calponin 3 was found to be a phosphoprotein (Online Figure IV), whereas MFG-E8 (Figure 1A and 1B), ␣-1-inhibitor III (data not shown), kininogen 1 (data not shown), and periostin (Online Figures V and VI) were detected as N-linked glycoproteins. Bioinformatic Functional Analysis of Differentially Abundant Age-Associated Arterial Proteins To identify potential roles of differentially abundant proteins with aging, proteins were grouped into various functional classes based on Swiss-Prot protein function and gene ontol- 1340 Circulation Research June 19, 2009 Downloaded from http://ahajournals.org by on June 14, 2020 Figure 1. Quantification and characterization of MFG-E8 by 2DIDE and iTRAQ in aging rat aorta. A, Enlarged region of 2D DIGE gel map showing that MFG-E8 is more abundant in aged aorta than young adult samples (2.53⫾0.15, n⫽8). Three solid white arrows point to gel spots identified as MFG-E8. Identifications were performed using light chromatography–MS/MS. B, iTRAQ MS spectra showing that MFG-E8 is more abundant in aged than young adult aorta. Representative MS spectra of 2 peptides VLPLSWHNR (409 – 417) and CLVTEDTQR (79 – 87) indicate the abundance of MFG-E8 is ⬇2.5-fold increased. ogy, and if needed, literature search (Table),15,16 with proteins being clustered into a number of cellular pathways, including apoptosis/cell cycle/proliferation, cytoskeleton/invasion, extracellular matrix/cell adhesion, metabolism, etc (Table). Notably, the majority of those proteins were related to the aging process or to the Ang II signaling cascade in other organs, ie, prostate, skeletal muscle, skin, etc (see the expanded Discussion section in the online data supplement), but many had not been previously linked specifically to arterial aging.7–14 Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) shows that the primary pathway was cellular movement, where 14 of these proteins could be linked together (Online Figure VII, A). The proteins include MFG-E8, apolipoprotein E, insulin-like growth factor binding protein 7, MMP2, biglycan, calponin 1, clusterin, collagen type I ␣ 1 chain, collagen type 1 ␣ 2 chain, fibronectin, periostin, serine protease HTRA1, transforming growth factor (TGF)-␤3, and vitronectin. The proteins increase in abundance in the old aorta. Together with Ang II, MCP-1, nuclear factor ␬B, PDGF, and other molecules, the proteins form a network of cellular movement, a salient feature of invasion (Online Figure VII, A). Interestingly, MFG-E8, a protein that markedly increased 2.3-fold in abundance with aging, is shown to be directly linked to protein kinase AKT17,18 and extracellular signal-regulated kinase 1/218 in this network (Online Figure VII, A). MFG-E8 is a multifunctional glycoprotein originally found in milk and mammary epithelial cells.18 –27 It is of particular interest because its internal fragment, known as medin, is an essential component of amyloid plaque deposition in aged human arteries.25,26 Its involvement in this cellular movement network indicates it may play an important role in regulating VSMCs invasive capability. Specifically, links between MFG-E8 and key molecules in vascular remodeling, such as Ang II and MCP-1, have not been established. We focused on MFG-E8 to determine Fu et al MFG-E8 Mediates VSMC Invasion 1341 how it specifically relates to the cell invasion signaling axis. Transcriptome, Immunolocalization, and Posttranslational Modification Analysis of MFG-E8 Using quantitative RT-PCR, MFG-E8 mRNA levels were increased 2.7-fold in older compared to younger rat aorta (Figure 2A; P⬍0.05). Western blotting analyses of 1DE (data not shown) and 2DE (Figure 2B) confirmed the increased abundance of MFG-E8 in older versus younger aorta. In addition to changes in the abundance of MFG-E8 with aging, 3 arterial MFG-E8 spots with similar molecular weight (MW) were observed in the 2DE of the sample from old aorta (Figure 2B). Importantly, treatment of the tissue samples with PNGase F, which specifically removes N-linked carbohydrates, shifted the triplicate spots to a lower MW, as anticipated for posttranslational modification by deglycosylation (Figure 2D). The newly appeared spots were identified as MFG-E8 by mass spectrometry. Immunofluorescence staining demonstrated that the intensity of glycosylated MFG-E8 increases within the old compared to the young aortic wall, and that MFG-E8 staining colocalizes with that of ␣ smooth muscle actin (␣-SMA), a marker of VSMCs (Figure 2C). Age-Associated Increase in MFG-E8 Expression Is Conserved Across Species Downloaded from http://ahajournals.org by on June 14, 2020 To determine whether the age-associated increase in MFG-E8 expression is conserved across various mammalian species, aortic tissues from nonhuman primates and humans were used for Western blotting and immunostaining studies. In nonhuman primates, Western blot analysis shows that the abundance of aortic MFG-E8 increases ⬇9.0-fold with aging, with the molecular weight indicating that it is glycosylated (Figure 3A). Immunolabeling analysis indicates that MFG-E8 staining (brown color) markedly increases in both the intima and the media aortic wall in older versus younger monkeys (Figure 3B). Similarly, in humans, Western blotting analysis and dual immunofluorescence staining of MFG-E8 indicate an age-associated increase ⬇6.5-fold of the glycosylated form of MFG-E8 (Figure 3C and 3D), predominantly located in the inner media and intimae and also colocalized with ␣-SMA, as in the rat and monkey (Figure 3C and 3B). Exogenous Administration of Ang II to Young VSMCs Mimics Aging by Elevating MFG-E8 Production Although bioinformatic functional analysis indicates that MFG-E8 is involved in a signaling network of cell movement (Online Figure VII, A), it fails to place it specifically downstream of the Ang II signaling cascade, a central feature of arterial aging.7–14 Dual immunofluorescence demonstrates that both Ang II and MFG-E8 expression within VSMCs are closely associated and that both markedly increase within the rat aortic wall, particularly in the thickened aged intima (Figure 4A). Its negative control is provided in Online Figure VIII. We used early passage VSMCs to detect a potential functional link between Ang II and MFG-E8. Both glycosy- Figure 2. Validation and location of the MFG-E8 expression within the rat aortic wall. A, Average MFG-E8 transcriptome (n⫽4). B, 2DE Western blot of MFG-E8. C, Immunofluorescence staining for MFG-E8 (green) and ␣-SMA (red). Counterstaining nuclei with a DAPI dye (blue). Merged image shows staining overlap (yellow) (original magnification, ⫻200). D, Enlarged region of DIGE 2DE showing PNGase F–treated MFG-E8 compared to an untreated endogenous sample (red) from the old rat aorta. PNGase F treatment (green) results in the removal of N-linked carbohydrates, shifting the spot position to a lower molecular weight. 1342 Circulation Research June 19, 2009 Figure 3. The age-associated arterial MGF-E8 increase is conserved in mammalian species including humans. A, Representative Western blots of monkey aortic MFG-E8 (left) and average data (right) (n⫽4). *P⬍0.05, old vs young. B, Immunohistostaining for MFG-E8 (brown color) within the monkey thoracic aortic wall (original magnification, ⫻100). L indicates lumen; M, media. C, Representative Western blots of human aortic MFG-E8 (left) and average data (right) (n⫽5). *P⬍0.05, old vs young. D, Double immunofluorescence staining for MFG-E8 (green) and ␣-SMA (red) and merged image (yellow) within the human thoracic aortic wall (original magnification, ⫻100). L indicates lumen; M, media. Downloaded from http://ahajournals.org by on June 14, 2020 lated (upper bands) and unglycosylated forms (lower bands) of MFG-E8 protein (Figure 4B, top) were detected, and both increased with aging in early passage VSMCs (Figure 4B, bottom).27 Treatment of young VSMCs with Ang II induced MFG-E8 production, predominantly in the native form (unglycosylated) in a dose-dependent manner, up to the levels of old untreated VSMCs (Figure 4B, bottom). Importantly, elevated MFG-E8, including glycosylated and unglycosylated forms, in old cells is significantly reduced (⬇40%) by treatment with [Sar1, Gly8]-Ang II acetate hydrate, an Ang II type 1 receptor blocker (Online Figure IX). This novel finding indicates that MFG-E8 is a downstream molecule of Ang II initiated signaling. MFG-E8 Stimulates Functional MCP-1 Production in Aging VSMCs and Consequently Enhanced Invasion Previous studies indicate that chronic infusion of Ang II to young rats increases MCP-1 expression and that the acute exposure of early passage VSMCs from young rat aortae potentiates their invasive capacity, in part, via enhanced MCP-1 expression.10,28,29 We probed for a relationship between MFG-E8 and MCP-1. Dual immunohistostaining of old aortae demonstrated MFG-E8 colocalization with MCP-1, preferentially in the thickened intima (Figure 5A and in Online Figure X), and immunocytostaining of old VSMCs indicates that MFG-E8 also colocalizes with MCP-1, preferentially in the perinuclear region (Figure 5B). MFG-E8 treatment of early passage VSMCs increased the functional dimer product of MCP-1 (Figure 5C).10,30 Conversely, MFG-E8 silencing (⬎70%) substantially reduces MCP-1 expression and its dimerization within VSMCs but does not effect MCP-1 mRNA levels (Online Figure XI, A and B). Furthermore, the Ang II– induced increase in MCP-1 expression is markedly inhibited by MFG-E8 silencing (Online Figure XI, C). Notably, the results in Figures 5 and 6 suggest that Ang II is upstream of MFG-E8, and that MCP-1 is downstream of MFG-E8 within Fu et al Downloaded from http://ahajournals.org by on June 14, 2020 Figure 4. Interplay between Ang II and MFG-E8 in vivo and in vitro. A, Immunofluorescence staining for MFG-E8 (green) and Ang II (red) and merged image (yellow) in rat aortic wall. Nuclei counterstaining with DAPI (blue) (original magnification, ⫻400). L indicates lumen; M, media. B, Representative 1DE Western blots of MFG-E8 in smooth muscle cells isolated from young or old rat aorta with or without Ang II treatment with indicated concentration levels (upper images). The MFG-E8 immunoreactivity doublet is the N-linked glycosylated form (upper bands) and unglycosylated (lower bands), as shown by deglycosylation of the sample with PNGase F resulting in enhancement of the lower molecular weight band. Average data (lower graph). *P⬍0.05 old untreated and young treated cells vs young control cells. the Ang II signaling cascade that potentiates VSMCs invasion. These data establish novel interactions among Ang II, MCP-1, and MFG-E8 (Online Figure VII, B). Next, we determined whether MFG-E8 is required for enhanced VSMCs invasion and whether it acts in this respect via MCP-1. Modified Boyden chamber analysis (Figure 6A, left) shows that, in control, VSMC invasive capability significantly was increased in old compared to young cells. MFG-E8 siRNA successfully knocked down MFG-E8 protein expression in both young and old VSMCs (Figure 6A, right) and reduced the invasive capability of both young and old VSMCs, eliminating age differences in invasive capacity that were present in control (Figure 6A, left). Exposure of VSMCs to Ang II increased the invasive capability of VSMCs in an age-dependent fashion, and this increase was substantially reduced by MFG-E8 silencing (Figure 6B). The addition of exogenous MFG-E8 also increased VSMCs invasive capability in an age-dependent fashion (Figure 6C), and these effects were substantially reduced by treatment with a specific MCP-1 receptor blocker vCCI (Figure 6C). Discussion In this study, we present for the first time a proteome profile of arterial aging in FXBN rats, a well-characterized rodent MFG-E8 Mediates VSMC Invasion 1343 Figure 5. Interaction between MFG-E8 and MCP-1 in vivo and in vitro. A, Immunofluorescence staining for MFG-E8 (green) and MCP-1 (red) and merged image (yellow) in old human aorta. Nuclei counterstaining with DAPI (blue) (original magnification, ⫻200). L indicates lumen; M, media. B, Immunofluorescence photomicrographs (⫻400) for MFG-E8 (red color), MCP-1 (green color), nuclei (blue color), and colocalization of MFG-E8 and MCP-1 (yellow color, right images) within VSMCs. C, Representative Western blots show that the functional dimer of MCP-1 increases in VSMC treated with MFG-E8 in both young and old cells in a dose-dependent manner. model with similarities to the human anatomic and physiological phenotype. Using a combination of 2DE and iTRAQ, we identified and characterized 923 proteins within the aortic wall, of which 50 significantly change abundance with aging, some of which also have posttranslational modifications such as phosphorylation and glycosylation. The proteins with significant abundance changes (Table) observed in this study play significant roles in a number of cellular pathways, including cellular movement, cell proliferation and apoptosis, energy metabolism, etc, and may contribute significantly to vascular remodeling with aging. Some of these proteins have been related to aging in other organs, ie, prostate, skeletal muscle, and skin, etc (for an expanded discussion regarding the proteins, see the online data supplement). Only a few proteins have been specifically linked to arterial aging, such as MMP2 or TGF-␤.7–14 These proteins, including Ang II, MCP-1, MFG-E8, MMP2, and TGF-␤, etc, are involved in a network of cellular movement (Online Figure VII, A). Furthermore, the present results establish molecular links between Ang II, MFG-E8, and MCP-1 in aged VSMCs. It has been previously established that Ang II and Ang II type 1 receptor expression and signaling increase within the arterial wall with aging.3,8,9,12,14 Exogenously infused Ang II to young animals produces the structural molecules of the arterial wall resembling old untreated animals,9,12 whereas chronic blockade of Ang II signaling in animals reverses and retards these 1344 Circulation Research June 19, 2009 Downloaded from http://ahajournals.org by on June 14, 2020 Figure 6. VSMC invasion assay. A, Average invasion analysis of VSMCs with and without siRNA of MFG-E8 (left graph). *P⬍0.05, old vs young cells chemoattracted by PDGF (10 ng/mL); #P⬍0.05, young and old cells with MFG-E8 silencing vs sham silencing, respectively. Representative Western blots show that the majority of MFG-E8 protein is knocked down via siRNA approach (right). B, Average invasion analysis of VSMCs with and without siRNA of MFG-E8 and Ang II treatment. *P⬍0.05, old vs young cells with treated with Ang II (100 nmol) and chemoattracted by PDGF (10 ng/mL); #P⬍0.05, young and old cells with MFG-E8 silencing vs sham silencing, respectively. C, Average invasion analysis of VSMCs treated with and without MFG-E8 plus vCCI. *P⬍0.05 young and old cells treated with MFG-E8 (50 ng/mL) vs untreated controls, respectively; #P⬍0.05, young and old cells treated with MFG-E8 plus vCCI vs MFG-E8 treated, respectively. age-associated structural molecular changes.31–33 Ang II can promote expression of MCP-1,10,28,29 a potent chemokine that is an element of the AKT-associated proinflammatory network and plays an important role in chemotaxis and atherosclerosis.34 Interestingly, MFG-E8 stood out in our proteomic analyses because it had not previously been linked directly with Ang II/MCP-1, VSMCs, or aging, although it has been shown to induce cell-specific apoptosis, metastasis, and angiogenesis.18 –24 Previous studies have shown that MFG-E8 is abundantly expressed in metabolically active tissues, ie, atherosclerotic plaques25,26,35 and tumors.24 We have shown that MFG-E8 is N-linked glycosylated and increases in abundance in aortae of old versus young rats (Table). This age-related increase in transcription and translation of MFG-E8 is preserved across species (rat, nonhuman primates, human), suggesting it may have a central role in arterial aging. As such, we investigated the interplay between MFG-E8 and Ang II, and for the first time, we uncovered the important finding that Ang II induces MFG-E8 in VSMCs isolated from rat aortae. In fact, MFG-E8 is required for the Ang II to increase MCP-1. Ang II was previously shown to coexpress with MCP-1.10,28,29 Furthermore, we show that Ang II or MCP-1 also colocalizes with MFG-E8 in the arterial wall, particularly in the aged thickened intimae. These findings suggest not only that MCP-1 receives Ang II signals but also that this cellular information is relayed via MFG-E8. Of note, treatment of isolated VSMCs with MFG-E8 induces the biologically active dimer of MCP-1 in a dose-dependent manner. This action of MFG-E8 is profound, because the active MCP-1 homodimer is a potent chemoattractant of smooth muscle cells, if produced locally.30 In addition, MFG-E8 enhanced the Ang II–induced invasion potential of the isolated VSMCs (Figure 6). We observed that the invasiveness of old VSMCs is greater than young cells and that this effect depends on Ang II signaling (Figure 6). However, silencing MFG-E8, a downstream molecule of Ang II signaling, similarly reduces the invasive capacity of both young and old cells, whereas exogenous MFG-E8 treatment of young VSMCs increases their invasiveness, up to the levels of old untreated cells. Interestingly, a CCR2 blocker, vCCI, can interrupt MFG-E8/MCP-1 signaling, sub- Fu et al Downloaded from http://ahajournals.org by on June 14, 2020 stantially inhibiting this effect. Thus, MFG-E8 is a novel link between Ang II/MCP-1 signaling and VSMCs invasive capacity within the aortic wall. Taken together, our study demonstrates a comprehensive nonbiased proteomic approach to better understand the biology of age-associated thickening of the aorta and has identified a novel Ang II/MFG-E8/MCP-1 signaling axis (Online Figure VII, B) that links the cellular movement signaling cascades with the age-associated increase in smooth muscle cell invasive capability. The abundance changes of other proteins observed in this proteomic study suggest that coordinated actions occur through this signaling axis. Notably, recent findings show that MFG-E8 is expressed in advanced human atherosclerotic plaques, and MGF-E8 deficiency results in the accumulation of apoptotic cells and accelerates atherosclerosis in mice.36 Previously, we have performed immunostaining for inflammatory cells, including monocyte/ macrophage and lymphocyte markers, and assessed apoptosis by TUNEL staining in the vasculature of young and old animals. Results have shown that these cells are not detected in both the intima and the media, rarely observed in the adventitia.11 Obviously, the regulation of these proteins is complex; however, based on the evidence mentioned above and provided in this report, it is likely that the accumulation of MFG-E8 in the arterial wall plays a critical role in arterial aging. Our data show MFG-E8 accumulates in the aorta with aging and promotes VSMC invasion, an important step in vascular thickening and arthrosclerosis. Further studies will show whether MFG-E8 can be a potential molecular target for the intervention to prevent/retard human arterial aging, arthrosclerosis, and other vascular diseases that involve inflammation and smooth muscle cell invasion. Acknowledgments We thank Robert O’Meally for technical support and Lesley Kane for critical reading of the manuscript. 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