S100A8 and S100A9 in Human Arterial Wall

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.

Atherosclerosis is a progressive chronic disease for which the prevailing "response-to-injury" hypothesis emphasizes endothelial dysfunction and accommodates the various risk factors, underscoring the importance of the immune system and chronic inflammation in its initiation, progression, and complications. Advanced atherosclerosis is often associated with dystrophic calcification, and there is a relationship between calcification and the severity of coronary disease. Dystrophic calcification results in less compliant and more rupture-prone vessels, precipitating ischemic and thrombotic complications. Initiation of calcification occurs within matrix vesicles (MVs) 4 (1), extracellular microstructures originating from budding of plasma membranes (2). Calcifying MVs contain high levels of Ca 2ϩ binding acidic phospholipids, particularly phosphatidylserine (PS) (2) and membrane phosphatases, which hydrolyze a variety of naturally occurring organic phosphoesters (3,4) ultimately forming nascent calcium phosphate mineral. The membrane-invested internal microenvironment protects the precurrent mineral nuclei in a pre-crystalline state, before conversion to hydroxyapatite (5). The presence of bone morphogenic proteins and non-collagenous bone matrix proteins including osteopontin, osteonectin, and matrix Gla protein (MGP) in atherosclerotic lesions indicates that vascular calcification is actively regulated (6,7), although mechanisms are unclear.
Inflammation-associated "calgranulins," S100A8 (MRP-8) and S100A9 (MRP- 14), members of the S100 family of Ca 2ϩ -binding proteins (8), are highly expressed in numerous inflammatory conditions, including giant cell arteritis (9), cystic fibrosis, rheumatoid arthritis, dermatoses, some malignancies, and autoimmune diseases (10,11). They are major cytosolic proteins in neutrophils and monocytes and found in subsets of macrophages at chronic inflammatory sites, but not in tissue macrophages (10,11). Monocytes infiltrating early atherosclerotic lesions, and macrophages within mature plaque of ApoE Ϫ/Ϫ mice were reported as S100A9 ϩ -S100A8 Ϫ (12). Intracellular S100A8 and S100A9 essentially regulate phagocyte migration through calcium and mitogen-activated protein kinase (MAPK) transduction pathways affecting the microtubular system (13). Murine (m) S100A8 is a potent leukocyte chemoattractant (14); mS100A8-elicited macrophages exhibit a "pro-atherogenic phenotype," expressing high levels of CD11b/CD18, Fc and scavenger receptors that accumulate acetylated low density lipoprotein (LDL) in vitro and in vivo, with cholesterol-ester profiles similar to those in human plaque and thus may contribute to potential foam cell (FC) development (15). Extracellularly, the S100A8-S100A9 complex (known as calprotectin) exerts antimicrobial activity (10), stimulates IL-8 production by airway epithelial cells (16), and transports arachidonic acid to endothelial cell (EC) targets affecting pathological responses in inflammation and atherosclerosis (10). S100A8 is secreted by appropriately activated macrophages (10) and is exquisitely sensitive to oxidation, particularly by peroxide and hypo-* This study was supported in part by a National Health and Research Council of Australia chlorite (HOCl/OCl Ϫ ) (17), and a role in antioxidant defense is proposed (18). HOCl from activated leukocytes generates inter-/intramolecular covalent cysteine-lysine sulfinamide bonds in S100A8 (17). HOCl is generated by myeloperoxidase (MPO), which co-localizes with macrophages (19) in human plaque where it oxidizes LDL to a proatherogenic form (20) and may also regulate proteases contributing to plaque rupture (21). Other S100s, such as S100A2 (22), are also implicated in cell responses to oxidative stress and S100B and S100A1 may link Ca 2ϩ -and redox-mediated signaling pathways, possibly by Ca 2ϩmodulated S100 cysteine thiol-glutathione covalent interactions (23,24). Intracellularly, the S100A8-S100A9 complex forms a scaffold for NADPH-oxidase components (10) and regulates casein kinase activity (25). This is the first report of calgranulins in macrophages and FC in human atheroma and in areas of vascular calcification. S100A8 and S100A9 mRNA and protein were also expressed by microvessels in areas of neovascularization. Monomeric and complexed forms of S100A9 were predominant in MVs isolated from human carotid artery and aorta suggesting a role in regulation of dystrophic calcification. We show high amounts of S100A8 and S100A9 complexes in plaque extracts and demonstrate the exquisite sensitivity, particularly of S100A8, to oxidation by HOCl, strongly suggesting it as a ready target for oxidation in vivo.
Carotid and Aortic Specimens-Aortic and carotid artery specimens containing atherosclerotic lesions and apparently normal mural areas, collected according to the Declaration of Helsinki, were obtained from 39 patients undergoing endarterectomy and 14 (aged 32-86 years) undergoing aortic reconstruction, respectively, at St Vincent's and Concord General Repatriation Hospitals, Sydney.
Arterial Tissue Extraction-Specimens from 11 endarterectomy patients were pulverized under liquid nitrogen. Samples of apparently normal carotid artery (taken from a 74-year-old male who died of chronic obstructive pulmonary disease; 25-h post-mortem delay) were similarly processed and because of the low protein content, pooled extracts from two different mural sections analyzed. Samples (ϳ500 mg) in PBS/10 mM EGTA (1.5 ml, pH 7.5) containing protease inhibitor mixture (Roche Applied Science, Mannheim, Germany) were sonicated for 5 min, rotated at 4°C for 2 h then centrifuged (1,600 ϫ g, 20 min, 4°C). Supernatants were precleared with caprine anti-rabbit IgG (ϳ1 g/ml, 1 h at 4°C) and protein A-Sepharose (ϳ25 l/500 l, 15 min, 4°C) or agarose-conjugated caprine anti-rabbit IgG (Sigma), centrifuged and ϳ25-30 g of total protein (bicinchoninic acid assay, Sigma) used for analysis. Because of the propensity of S100 proteins to form disulfide (30) and sulfinamide cross-linking (17) under oxidative conditions, five additional endarterectomy specimens were prepared in PBS containing 1 mM sodium azide (inhibits MPO that generates HOCl) (31) and 10 mM N-ethylmaleimide (NEM; derivatizes free thiols) (18).
The procedure for transmission electron microscopy (TEM) was adapted from a previous study (33). To quantitate the calcium and phosphorous content of MVs, suspensions (25 l) were dried at 22°C for 2 h and then mounted onto carbon disks coated with Balzes carbon fiber. Element content was assessed using a Cambridge S-360 scanning electron microscope (SEM) with Iridium software. The SEM was equipped with an EDS detector (1XRF Kevex with beryllium window) and 1-5 nA of beam current at 20 kV were used to acquire x-ray data. To confirm pyrophosphohydrolase activity, the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) assay was performed on duplicate samples as described (34). Results are expressed as nmol of p-nitrophenol formed/ min/mg MV protein. Protein concentrations were determined with the bicinchoninic acid assay.
Extraction of cholesterol and lipids from isolated MVs was performed as described (35). To determine the cholesterol content, membrane samples (1 ml) were mixed with methanol (2.5 ml) and extracted into hexane (5.0 ml) (36). Cholesterol was analyzed by reverse phase high performance liquid chromatography as described (37) and phospholipid content by the Bartlett assay (38).

RESULTS
S100B, S100A8, and S100A9 Expression in Plaque-Arteries lacking features of atherosclerotic transformation did not contain cells expressing S100A8 (Fig. 1A) or S100A9 (not shown) in the intima. Nerve endings in the adventitia were S100A1 ϩ /S100B ϩ in normal and diseased tissue, and some cells distributed irregularly throughout all layers of the arterial wall, and particularly frequent around neovessels, were S100A1-/S100B-and fascin-positive, identifying them as DC (28) (not shown).
All vessel segments affected by early-and late-stage atherosclerosis contained large numbers of irregularly distributed, intensely stained S100A8 ϩ FC and macrophages (Fig. 1, B and C), some of which were S100A8 ϩ -CD68 ϩ although the majority around neovessels were CD68 ϩ -S100A8 Ϫ . About 50% of neovascular EC were S100A8 ϩ (Fig.  1B). In intimal areas containing CD68 ϩ macrophages, many FC were S100A8 ϩ (Fig. 1C). S100A9 was present in FC and non-FC, particularly CD68 ϩ macrophages around neovessels, and in EC of all neovessels. The surrounding extracellular matrix (ECM) exhibited relatively intense S100A9 immunoreactivity (Fig. 1D). In situ hybridization indicated S100A8 (not shown) and S100A9 mRNA in FC, extravasating monocytes around neovessels and microvessels in areas of neovascularization (Fig. 1E). S100A8 or S100A9 were not detected in the luminal endothelium of arteries or the larger vasa vasorum (not shown).
Consecutive sections indicated no S100A8 or S100A9 expression in T lymphocytes or smooth muscle cells (not shown). Overlapping patterns of distribution of S100A8 and S100A9 were apparent in FC and non-FC (not shown), particularly CD68 ϩ macrophages around neovessels.
In particular, anti-S100A9 immunoreactivity was associated with early ( Fig. 2A) and large advanced calcified deposits (Fig. 2, B-E) that were surrounded by S100A9 ϩ cells, mainly macrophages (CD68 ϩ ) and some FC, some of which were closely attached to calcified deposits (Fig.  2C). These deposits consistently contained S100A9 in the ECM (Fig.  2E). S100A9 associated with calcified deposits was evident in all 11 carotid and 9 aortic specimens examined. Serial sections of the same specimens stained with anti-S100A8 showed markedly less reactivity; some S100A8 was associated with calcified deposits in 4 carotid arteries and 2 aortas (not shown).
Characterization of MV Isolates-TEM confirmed the heterogeneous structural appearance of MV isolates (Fig. 3, A-C). Size varied from 40 -1,200 nm; some had electron-lucent content (Fig. 3A), whereas others were filled with material of middle electron density or contained an electron dense core (Fig. 3B). Some MVs were clustered (Fig. 3C), and FIGURE 1. S100 proteins in normal (A) and atherosclerotic arterial wall (B-D), detected with anti-S100A8 (A-C), and -S100A9 (D). A, anti-S100A8 reactivity in a normal artery. B, typical anti-S100A8 reactivity in an area of neovascularization. Large arrows indicate S100A8 ϩ cells around a neovessel; small arrows show S100A8 ϩ EC. C, arrows show S100A8 ϩ FC (rose) of macrophage origin (CD68 ϩ , brown). D, S100A9 was located in EC (small arrows), inflammatory cells (large arrows) and covering the ECM (stars) in areas of neovascularization. E, in situ hybridization showing S100A9 mRNA in an area of plaque neovascularization. S100A9-mRNA ϩ EC (small arrows), macrophage-like cells and FC (large arrows) are indicated and scale bars included. DECEMBER 16, 2005 • VOLUME 280 • NUMBER 50 the membrane was absent in the contact zones suggesting that MVs coalesced. NTPPPH activity in MV isolates from 8 carotid specimens (mean: 315 Ϯ 173 nmol/min/mg protein) was similar to levels reported for human vascular MVs (32). The lipid composition of two MV isolates was analyzed. Total sterol was 762.8 Ϯ 23.3 nmol/mg protein and total phospholipid 745.5 Ϯ 141 nmol/mg protein (TABLE ONE). Cholesterol esters comprised ϳ14% of total sterols. HPLC analysis indicated that cholesteryl dodecanoate was the major ester (ϳ79%); ϳ9.5% was cholesteryl oleate, and ϳ2.3% was cholesteryl arachidonate.

S100 Proteins in Atherosclerosis
The mineral composition of carotid MV isolates (Fig. 3D), assessed by energy-dispersive x-ray microanalysis and SEM, indicated a Ca-to-P ratio of 0.66:1. Lower levels of sodium, sulfur, and chlorine were also present. In three isolates, the ratio ranged from 0.47-0.76:1 with a mean value of 0.66 Ϯ 0.17:1.
Western Blotting of MV Extracts-Strong anti-S100A9 immunoreactivity in 2 carotid MV extracts separated under reducing/non-reducing conditions is shown (Fig. 4A) and is representative of 12 of 12 atherosclerotic carotid and 5 aortic MV isolates. Components migrating with mass equivalent to S100A9 monomer (ϳ14 kDa) were prominent, and DTT-resistant components with masses of homodimers (S100A9 2 , ϳ28 kDa), trimers (S100A9 3 , ϳ42 kDa), and possibly S100A8-S100A9 heterodimer (ϳ24 kDa) were evident. The component migrating at ϳ56 kDa could represent S100A9 4 and was reduced in both samples suggesting disulfide-bonded complexes. The minor components (Ն 65 kDa) FIGURE 2. Co-localization of S100A9 (brown) with calcified deposits (violet) in early (A) and advanced (B-E) atherosclerotic plaques stained with anti-S100A9. A, S100A9 was localized intra-and extracellularly. Numerous S100A9 ϩ cells showed illdefined borders suggesting their destruction. B, low magnification micrograph showing S100A9 ϩ cells located in an intimal area containing large calcified deposits. C and D show details of B. In Fig. 2C, some S100A9 ϩ cells (arrows) are closely attached to calcified deposits (asterisk). E, S100A9 ϩ cells exhibiting ill-defined contours (straight arrows) apposed with calcified deposits (asterisk); anti-S100A9 immunoreactivity is evident extracellularly (open arrow).

S100 Proteins in Atherosclerosis
observed following reduction may represent higher complexes derived from proteins that did not enter the gel. In contrast to S100A9, S100A8 monomer levels in MV were less intense, and high interspecimen variability was obvious (Fig. 4B), which was not seen with anti-S100A8-immunoreactive components in whole carotid extracts (Fig. 5). M1 and M2 contained higher amounts of monomer (ϳ10 kDa) relative to the low levels of S100A8 homodimer (ϳ20 kDa, S100A8 2 ) and S100A8-S100A9 heterodimer; 6 of 10 isolates had similar patterns. MV isolates from 4 carotid samples lacked S100A12 (lane 2, Fig. 4C), or S100A1/S100B (lane 5, Fig. 4C), other S100 proteins expressed in atherosclerotic lesions (28), suggesting a role, particularly for S100A9 in dystrophic calcification. S100A8 and S100A9 in Arterial Extracts-Preliminary characterization of the structural properties of S100s in plaque indicated compo-nents with masses equivalent to S100A8 (10 kDa) and S100A8 homodimers, in 15 of 16 specimens (S1-S16; four of the most representative are presented in Fig. 5). Other prominent complexes migrated at 24, 30, 34, 40, and 48 kDa and may represent S100A8-S100A9, S100A8 3 , S100A8 2 -S100A9, S100A8-S100A9 2 , and S100A8 2 -S100A9 2 . Some extracts with high amounts of monomer generally had lower levels of the complexes (24 and 34 kDa) and some lacked monomer, indicating interspecimen variability (not shown). Anti-OxS100A8, which reacted more strongly with HOCl-oxidized S100A8 (compare Figs. 5, G and F), detected similar components (Fig. 5, C and D) but was more reactive with the complexes than anti-S100A8 (Fig. 5, A and B). Although reduced samples contained higher oligomers, reduction did not increase levels of monomeric S100A8, suggesting DTT-resistant oxidative modifications such as dityrosine and sulfinamide cross-links. The complexes migrating at ϳ60 kDa following reduction may be derived from larger oligomers that failed to enter the gel, or from complexes that were less reactive with the antibody.
Apparently normal regions of artery did not contain cells expressing S100s (Fig. 1A), and extracts contained no monomeric S100A8 (Fig. 5E), and insignificant levels of monomeric S100A9 (Fig. 6C) were detected by anti-OxS100A8 and anti-S100A9, respectively. Low reactivity with components migrating at ϳ40 and ϳ60 kDa was observed with both antibodies; reduction decreased levels of the 60-kDa complex. Blood neutrophils express high amount of the S100s and represent another potential contaminating source. Although neutrophil lysates contained both protein monomers, no complexes were observed (Figs. 5E and 6C). S100A8-S100A9 cross-linked complexes were eluted from anti-S100A9-Sepharose (Fig. 6, D and E). DTT-resistant S100A8-S100A9 heterodimer (24 kDa), S100A9 2 (28 kDa), and the S100A9 2 -S100A8 complex (40 kDa) were detected using both antibodies. The failure to elute larger complexes may indicate that aggregates either fail to bind, or cannot be eluted from the column. Elution of S100A8 (10 kDa) also indicated the likelihood of non-covalent association that commonly occurs between S100A8 and S100A9 (10).

DISCUSSION
S100 proteins are emerging as important mediators of diverse processes in chronic inflammation. S100A1/B is found in DC associated with T lymphocytes, and may represent antigen-presenting cells in plaque (28). S100A9-and S100A12-positive monocytes reported in the ascending aortae of ApoE Ϫ/Ϫ mice (12,40) were suggested to regulate monocyte transmigration and endothelial activation even though there are no sequences related to human S100A12 within the rat and mouse genomes (8). Key cytokines involved in initiation and maturation of atheromatous plaque up-regulate S100 genes in numerous cell types. IL-1 induces strong S100A8 and S100A9 expression in murine microvascular EC (41) and in elicited murine macrophages (42). Here we demonstrate high expression of S100A8 and S100A9 in human plaque but not normal intima (Figs. 1A, 5E, and 6C), with distribution distinct from DC expressing S100A1/B (28). S100A8 and S100A9 were expressed in macrophages and FC although some CD68 ϩ cells were FIGURE 5. Western blot of plaque extracts from 5 patients (S1-S5) using anti-S100A8 (A and B), anti-OxS100A8 (C and D), normal arterial extracts (E), and ELISA assessing reactivities of both antibodies (F and G). A and B, reactivity of anti-S100A8 with S100A8 and plaque extracts under non-reducing (A) and reducing (B) conditions. Relative positions of S100A8 (A8, ϳ50 ng), S100A8 2 (A8 2 ) and MW markers are indicated. C and D, reactivity of anti-OxS100A8 with S100A8 (ϳ50 ng) and plaque extracts separated under non-reducing (C) and reducing (D) conditions (DTT levels given). Anti-OxS100A8 reacted more strongly with the complexes than anti-S100A8. Non-reduced samples were run simultaneously on another gel. E, normal arterial extract (N) and neutrophil lysates (PMN; 2 l, 10 5 cells/ml) were separated with/without DTT (100 mM) and blotted with anti-OxS100A8; migration of monomeric S100A8 (A8) is indicated. F and G, reactivities of anti-S100A8 (F) and anti-OxS100A8 (G) with recombinant S100A8 (Ⅺ) and OxS100A8 (f) using a double-sandwich ELISA system (27). Anti-S100A8 (F) or anti-OxS100A8 (G) was used for capture and detection of 0.0625-1.5 nM S100A8 or OxS100A8 (abscissa) and absorbance measured at 405 nm (y-axis). Anti-S100A8 reacted similarly with S100A8 and OxS100A8 (F); anti-OxS100A8 reacted more strongly with OxS100A8 than with S100A8 (G). negative (Fig. 1C), suggesting differential activation of macrophage populations, similar to the heterogeneity of MPO-expressing macrophages seen in plaque (19). Expression of S100A8 contradicts findings in murine plaque where only S100A9 was found, and questions the proposal that the absence of S100A8 would favor atherogenesis because of its ability to negate S100A9-enhanced leukocyte adhesion (12). Importantly, intracellular S100A8 and S100A9 essentially regulate phagocyte migration by integrating the calcium and MAPK transduction signals, thereby controlling reorganization of the phagocyte microtubular system (13) and monocytes expressing membrane-bound S100A8/S100A9 secrete more TNF and IL-1 than their surface negative counterparts (43) thus potentially favoring atherogenesis.
All neovessels, but not larger vessels, were strongly S100A9 ϩ / S100A8 ϩ (Fig. 1). Expression of S100A8/S100A9 by EC is controversial. Others suggest that adherent myeloid cells release the proteins and S100A9 may bind to venules via heparan sulfate glycosaminoglycans (44) and that this process triggers EC activation (45). Although this may also be the case, we showed up-regulation of the two genes in three microvascular endothelial cell lines by IL-1/TNF (41). Here we demonstrate S100A8 and S100A9 mRNA (Fig. 1E) in neovessels, strongly supporting our proposal that they are expressed by appropriately activated microvessels, but not larger vessels, and may regulate leukocyte migration in the microcirculation (41). S100A9 was more prominent than S100A8 on the surrounding ECM (Fig. 1D) and in areas of early and advanced calcification (Fig. 2), possibly because of its strong heparin affinity. S100A8 and S100A9 may be involved in bone remodeling. 5 Here we show S100A9 ϩ cells with ill-defined contours apposed to calcified deposits in intima (Fig. 2); little S100A8, but high levels of S100A9 monomer and complexes were found in enzymatically active MV extracts (Fig. 4), suggesting S100A9 involvement in mural dystrophic calcification. In contrast, MV extracts lacked S100A12, which is also expressed FIGURE 6. Western blots showing anti-S100A9 immunoreactivity with plaque extracts (A and B) or normal extracts (C) and detection of S100A8 and S100A9 by anti-S100A9-immunoaffinity (D and E). A and B, extracts separated with (A) and without (B) DTT (100 mM) were blotted with anti-S100A9. Arrows indicate S100A9 standard (A9, ϳ50 ng) and MW markers. C, normal arterial extract (N) and neutrophil lysates (PMN; 2 l, 10 5 cells/ml) separated with/without DTT (100 mM); migration of monomeric S100A9 (A9) is indicated. D and E, Western blot analysis (Ϯ100 mM DTT) of immunopurified plaque extracts. Eluates from immobilized anti-S100A9 (lanes 1 and 3) and S100A8 (A8) or S100A9 (A9) standards (lanes 2 and 4) were blotted with anti-S100A9 (D) or anti-S100A8 (E). S100A8-S100A9 complexes were detected with both antibodies. Note DTT-resistant complexes.  11), and S100A8 (ϳ40 ng, lanes 4 and 8; 900 ng, lane 12) immunoblotted with anti-OxS100A8, anti-S100A8, or HOP-1. B, S100A8 (A8, ϳ40 ng) and C, S100A9 (A9, ϳ40 ng), oxidized with increasing molar excess HOCl and separated under reducing conditions. Note the dimers and complexes (arrows) resistant to DTT (100 mM).
in macrophages and FC in plaque, 6 and S100B, which is exclusively found in DC within atheromatous lesions (46). There is emerging evidence for a role for S100 proteins in calcification/bone remodeling. Calbindin-D9k, is also present in MVs and enhances mineralization and interstitial Ca 2ϩ absorption (47). S100A6 stimulates osteoblast proliferation, expression of alkaline phosphatase and promotes osteoblastogenesis in response to simvastatin, which stimulates bone formation (48), whereas, S100A4 may inhibit mineralization, possibly by modulating osteoblast differentiation (49). Other high affinity Ca 2ϩ -binding proteins, including osteoprotegerin, MGP, and fetuin protect against dystrophic calcification (50).
The Ca:P ratio in MV isolates (Fig. 3D) was 0.66:1.0, which is less than the Ca:P ratio of 1.66:1, reported for vascular hydroxyapatite (51). However, MV isolates were structurally heterogeneous (Fig. 3), and high concentrations of phosphate indicate incomplete mineralization, which occurs when phosphate esters localize to the MV membrane before hydroxyapatite forms (52). The MVs also contain high amounts of phospholipids, thereby elevating total P i levels (53) and local concentrations of P i increase by enzymes such as the ATPases and NTPPPH (47). Osteopontin and MGP inhibit vascular calcification within atherosclerotic lesions (54,55) and phosphorylated osteopontin (56), and its phosphopeptides inhibit hydroxyapatite formation (57). S100A8 and S100A9 can inhibit casein kinase activity (25) and S100A9 is phosphorylated at Thr 113 in activated monocytes and neutrophils (58,59). Although the kinases responsible for S100A9 phosphorylation are unclear, phosphorylated isoforms of S100A9 translocate to the plasma membrane and cytoskeleton and exhibit increased Ca 2ϩ binding compared with the non-phosphorylated isoforms, suggesting that phosphorylation modulates Ca 2ϩ binding and membrane translocation (60).
Lipid analysis of human vascular MV isolates has not been reported. These contained approximately equimolar amounts of phospholipids and sterols of which 2.3% comprised cholesteryl arachidonate (TABLE  ONE). This contrasts with chicken MVs, which contain mostly phospholipids with smaller amounts of free cholesterol, fatty acids, and triacylglycerols (2). During MV calcification, extensive phospholipid degradation occurs and levels of fatty acids increase. However, PS, which complexes to nascent mineral within the MV and is only extractable after demineralization, is not only protected from degradation but is apparently synthesized (61). The S100A8-S100A9 heterocomplex binds reversibly to unsaturated fatty acids in vitro, and it is the major arachidonic acid-binding protein in human neutrophils and may contribute to endothelial cell fatty acid uptake (10). S100A8 and S100A9 translocate into cholesterol-and sphingolipid-rich lipid rafts, other lipid structures and cell membranes in a detergent-resistant and Ca 2ϩ -and tubulin-dependent manner (62). Annexins are the main group of proteins in MVs; annexins A2, A5, and A6 facilitate MV Ca 2ϩ influx by forming ion channels (47). Annexin A5 binds PS in a Ca 2ϩ -dependent manner within the nucleational core of growth plate MVs stimulating crystal growth (2) and annexin A2 may facilitate mineralization in osteoblastic lipid rafts (63). Several S100 proteins interact with annexins (64,65) and considering the co-localization within MVs and common Ca 2ϩ -and lipid-binding properties of annexins and S100A8/S100A9, their interaction is worthy of investigation.
HOCl-oxidized proteins, particularly LDL, are found in plaque extracts (20). HOCl generated DTT-resistant S100A8 dimers and trimers in vitro within 10 min, with as little as equimolar, to 5-fold excess (Fig. 7B). Intrachain sulfinamide bonds predominate with low molar levels of HOCl but these cannot be identified by SDS-PAGE (17). S100A9 was somewhat less susceptible, requiring 5-20-fold excess HOCl (Fig. 7C). The rapid oxidation of these proteins with equimolar levels of HOCl contrasts markedly with those required to oxidize LDL or BSA (ϳ1:800, or ϳ1:100, respectively for 2 h (20) ; Fig. 7A) in vitro. OxS100A8 was not recognized by HOP-1, suggesting that levels of HOCl-modified proteins in plaque may be greater than earlier estimates (20). In addition to HOCl cross-linking LDL via chloramines, which can subsequently react with lysine or histidine residues (66), a previously unsuspected oxidation pathway via cysteine-lysine interactions was also proposed, with likely relevance in the pathogenesis of atherosclerosis (68). Our evidence strongly supports this possibility, particularly as MPO (69) and S100A9 (Figs. 1D and 2E) bind glycosaminoglycans on EC and the ECM, and S100A8 preferentially forms non-covalent complexes with S100A9 (10), so the close proximity of these proteins may initially reduce oxidative damage. S100A8 and S100A9 in atherosclerotic plaque and calcifying MVs may significantly influence redox-and Ca 2ϩ -dependent processes during the pathogenesis of atherosclerosis and its chronic complications particularly dystrophic calcification.