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According to articles I read, AGEs (advanced glycation end products) activate RAGEs (receptors for AGEs). This activation increases the ROS (reactive oxygen species) levels in the cells.
Free radicals can cause damage in cells, which is definitely not good. What do you think, what is the role of RAGEs, which is more important than cellular damage?
RAGE Signaling in Skeletal Biology
Purpose of review: The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease.
Recent findings: The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered.
Keywords: Bone Osteoblast Osteoclast Osteocyte Osteoporosis RAGE.
Conflict of interest statement
Lillian Plotkin, Alyson Essex and Hannah Davis declare no conflict of interest.
The Receptor for Advanced Glycation Endproducts [RAGE] is a member of the immunoglobulin superfamily, encoded in the Class III region of the major histocompatability complex [1–4]. This multiligand receptor has one V type domain, two C type domains, a transmembrane domain, and a cytoplasmic tail. The V domain has two N-glycosylation sites and is responsible for most (but not all) extracellular ligand binding . The cytoplasmic tail is believed to be essential for intracellular signaling, possibly binding to diaphanous-1 to mediate cellular migration . Originally advanced glycation endproducts (AGEs) were indeed thought to be its main activating ligands, but since then many other ligands of RAGE including damage-associated molecular patterns (DAMP's) have been identified [1, 7, 8]. RAGE is thus considered a pattern-recognition receptor (PRR), having a wide variety of ligands [9–11].
RAGE is expressed as both full-length, membrane-bound forms (fl-RAGE or mRAGE, not to be confused with mouse RAGE) and various soluble forms lacking the transmembrane domain. Soluble RAGE is produced by both proteolytic cleavage of fl-RAGE and alternative mRNA splicing. The soluble isoforms include the extracellular domains but lack the transmembrane and cytoplasmic domains [12–15]. Soluble RAGE derived specifically from proteolytic cleavage is sRAGE, although this terminology is not consistent in the literature – sRAGE sometimes refers to soluble RAGE in general. RAGE is expressed at low levels in a wide range of differentiated adult cells in a regulated manner but in mature lung type-I pneumocytes it is expressed at substantially higher levels than in other resting cell types. It is highly expressed in readily detectable amounts in embryonic cells . RAGE is also highly expressed and associated with many inflammation-related pathological states such as vascular disease, cancer, neurodegeneration and diabetes (Figure 1) [17, 18]. The exceptions are lung tumors and idiopathic pulmonary fibrosis, in which RAGE expression decreases from a higher level in healthy tissue [19, 20].
RAGE is Central to Many Fundamental Biological Processes. Focusing on RAGE allows us to view many aspects of disordered cell biology and associated chronic diseases. Chronic stress promotes a broad spectrum of maladies through RAGE expression and signaling, focusing the host inflammatory and reparative response.
RAGE and Soluble RAGE
Human RAGE mRNA undergoes alternative splicing, much as with other proteins located within the MHC-III locus on chromosome 6. A soluble form with a novel C-terminus is detected at the protein level, named "Endogenous Secretory RAGE" (esRAGE or RAGE_v1) . This form is detected by immunohistochemistry in a wide variety of human tissues that do not stain for noticeable amounts of fl-RAGE . Over 20 different splice variants for human RAGE have been identified to date. Human RAGE splicing is very tissue dependant, with fl-RAGE mRNA most prevalent in lung and aortic smooth muscle cells while esRAGE mRNA is prevalent in endothelial cells. Many of the splice sequences are potential targets of the nonsense-mediated decay (NMD) pathway and thus are likely to be degraded before protein expression. Several more lack the signal sequence on exon1 and thus the expressed protein could be subject to premature degradation. The only human variants that have been detected at the protein level in vivo is are fl-RAGE, sRAGE, and esRAGE [17, 22].
Human fl-RAGE is also subject to proteolytic cleavage by the membrane metalloproteinase ADAM10, releasing the extracellular domain as a soluble isoform [12–14]. Antibodies raised to the novel C-terminus of esRAGE do not recognize the isoform resulting from proteolytic cleavage. In serum the predominant species is the proteolytic cleavage and not mRNA splicing isoform . Enhancement of proteolytic cleavage will increase soluble RAGE levels, while inhibition will increase fl-RAGE levels. This cleavage process is modulated by Ca++ levels, and following proteolytic cleavage the remaining membrane-bound C-terminal fragment is subject to further degradation by γ-secretase [13, 14]. Cleavage of the C-terminal fragment by γ-secretase will release a RAGE intercellular domain (RICD) into the cytosolic/nuclear space. Even though RICD has not yet been detected and is presumably degraded quickly, overexpression of a recombinant form of RICD will increase apoptosis as measured by TUNEL assay, indicating RAGE processing has another intercellular role .
Murine fl-RAGE mRNA also undergoes alternative splicing, and some of the splice products are orthologs of esRAGE . To date over 17 different mRNA splices have been detected. As with human splice variants, mouse splice variants are expressed in a tissue-dependant fashion and many are targets of NMD. Several common splice patterns exist when comparing human and mouse RAGE, although variants that would give rise to a soluble isoform are much rarer in mice .
Recombinant RAGE has been cloned into a variety of expression vectors, and native soluble RAGE has been purified from murine, bovine, and human lung [24–28]. A recombinant soluble isoform takes on a dominant-negative phenotype and blocks signaling. Soluble RAGE can act as an extracellular "decoy receptor", antagonizing fl-RAGE and other receptors by binding DAMPs and other ligands and inhibiting leukocyte recruitment in a variety of acute and chronic inflammatory conditions . Both esRAGE and sRAGE act as decoy receptors for the ligand HMGB1 . However soluble RAGE has functions other than just blocking fl-RAGE function, and exerts pro-inflammatory properties through interaction with Mac-1 [10, 29]. Thus although soluble RAGE has protective properties in the setting of chronic inflammation, it might be better described as a biomarker of chronic inflammation [30, 12]. Information on long-term effects of treatment with exogenous soluble RAGE is still not available, and it has yet to be shown that plasma levels of soluble RAGE are sufficient to effectively act as a decoy receptor in vivo .
The two different properties of soluble RAGE (decoy receptor and pro-inflammatory) and the different pathways associated with its production might explain why there are both positive and negative correlations between its levels in human serum and disease. Total soluble RAGE in serum is significantly lower in non-diabetic men with coronary artery disease than those without . As assessed by delayed-type hypersensitivity and inflammatory colitis, soluble RAGE suppressed inflammation In IL-10 deficient mice, reduced activation of NFκB, and reduced expression of inflammatory cytokines [32, 33]. RAGE knockout mice have limited ability to sustain inflammation and impaired tumor elaboration and growth. Thus, RAGE drives and promotes inflammatory responses during tumor growth at multiple stages and has a central role in chronic inflammation and cancer .
Lower levels of soluble RAGE levels are found in Amyotrophic Lateral Sclerosis (ALS), and lower esRAGE levels predict cardiovascular mortality in patients with end-stage renal disease [35, 36]. In patients with type 2 diabetes higher soluble RAGE levels positively correlate with other inflammatory markers such as MCP-1, TNF-α, AGEs, and sVCAM-1 [37, 38]. Total soluble RAGE but not esRAGE correlates with albuminuria in type 2 diabetes . Interestingly, although changes in human serum levels of soluble RAGE correlate very well with progression of inflammation-related pathologies, in mouse serum soluble RAGE is undetectable . This contrasts the importance of splicing and proteolytic cleavage forms soluble RAGE in mice and humans . One caution is that although ELISA-based assays of soluble RAGE in serum show high precision and reproducibility, the levels show high variation (500–3500 ng/L P < 0.05) among otherwise healthy donors . Soluble RAGE levels correlate with AGE levels even in non-diabetic subjects . Thus, although one measurement of soluble RAGE may not be sufficient to predict a pathological state, changes in levels over time could be predictive of the development of a disease.
RAGE Signaling Perpetuates the Immune and Inflammatory Response
A recent review extensively covers the role of RAGE signaling in diabetes and the immune response . Activation of multiple intracellular signaling molecules, including the transcription factor NF-κB, MAP kinases, and adhesion molecules are noted following activation of RAGE. The recruitment of such molecules and activation of signaling pathways vary with individual RAGE ligands. For example, HMGB1, S100B, Mac-1, and S100A6 activate RAGE through distinct signal transduction pathways [42, 43]. Ann Marie Schmidt posited a "two-hit" model for vascular perturbation mediated by RAGE and its ligands . This "two-hit" model hypothesizes that the first "hit" is increased expression of RAGE and its ligands expressed within the vasculature. The second "hit" is the presence of various forms of stress (e.g. ischemic stress, immune/inflammatory stimuli, physical stress, or modified lipoproteins), leading to exaggerated cellular response promoting development of vascular lesions. Most importantly, engagement of RAGE perpetuates NF-kB activation by de novo synthesis of NF-kBp65, thus producing a constantly growing pool of this pro-inflammatory transcription factor . RAGE is associated with amplified host responses in several pathological conditions, including diabetes, chronic inflammation, tumors, and neurodegenerative disorders . We would similarly posit that during periods of epithelial barrier disruption that both signal 1, a growth factor stimulus, and signal 2, various forms of stress, in conjunction with RAGE and RAGE ligands helps mediate this effect.
RAGE ligands fall into several distinct families. They include the High Mobility Group family proteins including the prototypic HMGB1/amphoterin, members of the S100/calgranulin protein family, matrix proteins such as Collagen I and IV, Aβ peptide, and some advanced glycation endproducts such as carboxymethyllysine (CML-AGE) [4, 6, 16, 45]. Not all members of these families have been identified as RAGE ligands, and many RAGE ligands have a variety of RAGE-independent effects . AGE molecules are prevalent in pathological conditions marked by oxidative stress, generation of methoxyl species, and increases in blood sugar, as found in type 2 diabetes mellitus [6, 27]. The S100/calgranulin family consists of closely related calcium-binding polypeptides which act as proinflammatory extracellular cytokines.
Ligand accumulation and engagement in turn upregulates RAGE expression . It is not known why some ligands (such as HMGB1, some S100's, and CML-AGE) cause strong pro-inflammatory signaling through RAGE, while similar molecules (such as pentosidine-AGE and pyrraline-AGE) seem to have much less or no signaling. The most commonly accepted hypothesis to reconcile these differences involves ligand oligomerization. Of the identified RAGE ligands, those that oligomerize activate RAGE more strongly . Oligomers of ligands could potentially recruit several RAGE receptors as well as Toll-like receptors [TLRs] at the cell surface or at intracellular vesicles and induce their clustering on the cell surface. For example, S100 dimers and higher-order multimers bind several receptors including TLR4, and clustering of RAGE could promote a similarly strong response . Recent studies show that AGEs and certain S100 multimers will cluster RAGE in this manner [11, 48, 49]. However this does not completely explain why some ligands will activate RAGE strongly while structurally similar ones do not seem to activate it at all .
Overview of HMGB1 and the HMG Protein Family
HMG (High Mobility Group) proteins are very basic, nuclear, non-histone chromosomal proteins of which HMGB1 is the only member that has been shown to activate RAGE. The HMG proteins are not to be confused with the unrelated compound in the mevalonate pathway "HMG-CoA" (3-hydroxy-3-methylglutaryl coenzyme A) and "HMG-CoA reductase inhibitors" (statins) . The HMG proteins were first identified in calf thymus in 1973 and named for their high mobility in protein separation gels . Typically they have a high percentage of charged amino acids and are less than 30 kDa in mass. HMG proteins are expressed in nearly all cell types, relatively abundant in embryonic tissue, and bind to DNA in a content-dependant but sequence-independent fashion . They are important in chromatin remodeling and have many other functions. Mouse knockout data shows that the loss of any one of the HMG proteins will result in detectable deleterious phenotypic changes. Of those, the HMGB1 (-/-) mice die of hypoglycemia within 24 hours of birth [54, 55]. Extended back-crossing of the knockout allele into various murine strains have revealed an even more profound phenotype with mice dying by E15 of development [Marco Bianchi, personal communication]. The homology between mouse and human HMGB1 is extraordinary with only two amino acid differences observed. Similar profound homology exists throughout vertebrate species with 85% homology with zebrafish.
There are three sub-classifications of HMG proteins: HMGA, HMGB, and HMGN (Table 1). There is also a similar set known as HMG-motif proteins. The HMG-motif proteins differ in that they are cell-type specific, and bind DNA in a sequence-specific fashion. HMGA proteins (formerly HMGI/Y) are distinguished from other HMG proteins by having three AT-hook sequences (which bind to AT-rich DNA sequences) [56, 57]. They also have a somewhat acidic C-terminal tail, although the recently discovered HMGA1c has no acidic tail and only two AT-hooks. HMGN proteins (formerly HMG14 and HMG17) have nucleosomal binding domains. HMGB proteins (formerly HMG1 through HMG4) are distinguished by having two DNA-binding boxes that have a high affinity for CpG DNA, apoptotic nuclei, and highly bent structures such as four-way Holliday junctions and platinated/platinum-modified DNA. The HMGB proteins have a long C-terminal acidic tail except for HMGB4, which recently has been detected at the protein level in the testis where it acts as a transcriptional repressor . The HMGB acidic tail consists of at least 20 consecutive aspartic and glutamic acid residues. A C-terminal acidic tail of this length and composition is rarely seen in Nature, although a few other autophagy and apoptosis-related proteins such as parathymosin have a long internal stretch of acidic peptides [59–61].
Of the HMG proteins, HMGB1 has an additional cytosolic and extracellular role as a protein promoting autophagy and as a leaderless cytokine, respectively . Macrophages, NK cells and mature DCs actively secrete HMGB1, and necrotic cells passively secrete it. HMGB1 has also been detected in the cytosol, depending on the cell type, where it has a major positive role in regulating autophagy . Although HMGA1 has a role in the export of HIPK2 (Homeodomain-interacting protein kinase 2, a proapoptotic activator of p53) from the nucleus to the cytoplasm , the HMG proteins other than HMGB1 are very seldom detected outside the nucleus. This is likely explains why HMGB1 is the only member of the family that activates RAGE . Since HMGB1 translocates between the nucleus and cytosol, there is a possibility that it could bind to soluble RAGE in the cytosol and thereby play a role in regulating its activity.
Biochemistry of HMGB1
HMGB1 is a highly conserved protein consisting of 215 amino acids. It is expressed in almost all mammalian cells. Human HMGB1 shares an 80% similarity with HMGB2 and HMGB3 . It has two lysine-rich DNA binding boxes (A- and B-) separated by a short linker. The boxes are separated from the C-terminal acidic tail by another linker sequence ending in four consecutive lysines. An isoform believed to result from cleavage of the acidic tail has been detected in vivo . HMGB1 has three cysteines, of which the first two vicinal cysteines (Cys 23 and 45, based on Met1 as the initial Met in the immature protein) can form an internal disulfide bond within the A-box. The A-box and the oxidation state of these two cysteines play an important role in the ability of HMGB1 to bind substrates. Oxidation of these two cysteines will also reduce the affinity of HMGB1 for CpG-DNA [67, 68]. Addition of recombinant A-box antagonizes HMGB1's ability to bind other substrates [67, 69]. It remains to be determined if the action of the A-box is the result of competitive inhibition by binding to other substrates or interfering with the ability of the B-box to bind substrates. The two boxes acting in concert will recognize bent DNA . The third cysteine (Cys106, in the B-box) often remains reduced and is important for nuclear translocation . The region around this cysteine is the minimal area with cytokine activity . HMGB1 undergoes significant post-translational modification, including acetylation of some lysines, affecting its ability to shuttle between the nucleus and cytosol [71, 72]. DNA-binding and post-translational modification accessibility can be modulated by interactions of the acidic tail with the basic B-box [73–75]. HMGB1 signals through TLR2, TLR4, and TLR9 in addition to RAGE [76, 77]. It also binds to thrombomodulin and syndecan through interactions with the B-box .
Evolution of HMGB1
HMG proteins can be found in the simplest multi-cellular organisms . The two DNA boxes resulted from the fusion of two individual one-box genes . The two-box structure makes it particularly avid specific for bent DNA, and is highly conserved among many organisms [81, 82]. This similarity makes generation of HMGB1-specific antibodies a challenge. Antibody cross-reactivity could result from the strong similarity of HMGB1 across individual species, HMGB1 to other HMGB proteins, and even HMGB1 to H1 histones (Sparvero, Lotze, and Amoscato, unpublished data). The possibility of misidentification of HMGB1 must be ruled out carefully in any study. One way to distinguish the HMGB proteins from each other is by the length of the acidic tail (30, 22, and 20 consecutive acidic residues for HMGB1, 2, and 3 respectively, while HMGB4 has none). The acid tails are preceded by a proximal tryptic cleavage site, and they all have slightly different compositions. This makes mass spectrometry in conjunction with tryptic digestion an attractive means of identification.
Normal/healthy levels of HMGB1
Relative expression of HMGB1 varies widely depending on tissue condition and type. Undifferentiated and inflamed tissues tend to have greater HMGB1 expression than their counterparts. Spleen, thymus and testes have relatively large amounts of HMGB1 when compared to the liver. Subcellular location varies, with liver HMGB1 tending to be found in the cytosol rather than the nucleus [55, 83]. HMGB1 is present in some cells at levels exceeded only by actin and estimated to be as much as 1 × 10 6 molecules per cell, or one-tenth as abundant as the total core histones. But this number should be regarded with some caution since it includes transformed cell lines and does not define the levels of HMGB1 abundance in vivo in most cellular lineages . The levels of serum HMGB1 (as determined by Western Blot) have been reported with wide ranges: 7.0 ± 5.9 ng/mL in healthy patients, 39.8 ± 10.5 ng/mL in cirrhotic liver and 84.2 ± 50.4 ng/mL in hepatocellular carcinoma . For comparison, human total serum protein levels vary from about 45–75 mg/mL, and total cytosolic protein levels are about 300 mg/mL [85, 86]. This puts serum HMGB1 in the low part-per-million range by mass, making detection and separation from highly abundant serum proteins challenging.
HMGB1 and RAGE in cancer and inflammation
HMGB1, along with RAGE, is upregulated in many tumor types (Table 2). HMGB1 is passively released from necrotic cells but not from most apoptotic cells. The reason for this is unknown, but has been hypothesized to be a result of either redox changes or under-acetylation of histones in apoptotic cells [87, 88]. HMGB1(-/-) necrotic cells are severely hampered in their ability to induce inflammation. HMGB1 signaling, in part through RAGE, is associated with ERK1, ERK2, Jun-NH2-kinase (JNK), and p38 signaling. This results in expression of NFκB, adhesion molecules (ICAM, and VCAM, leading to macrophage and neutrophil recruitment), and production of several cytokines (TNFα, IL-1α, IL-6, IL-8, IL-12 MCP-1, PAI-1, and tPA) . An emergent notion is that the molecule by itself has little inflammatory activity but acts together with other molecules such as IL-1, TLR2 ligands, LPS/TLR4 ligands, and DNA. HMGB1 signaling through TLR2 and TLR4 also results in expression of NFκB. This promotes inflammation through a positive feedback loop since NFκB increases expression of various receptors including RAGE and TLR2. LPS stimulation of macrophages will lead to early release of TNFα (within several hours) and later release of HMGB1 (after several hours and within a few days). Targeting HMGB1 with antibodies to prevent endotoxin lethality therefore becomes an attractive therapeutic possibility, since anti-HMGB1 is effective in mice even when given hours following LPS stimulation . HMGB1 stimulation of endothelial cells and macrophages promotes TNFα secretion, which also in turn enhances HMGB1 secretion . Another means to induce HMGB1 secretion is with oxidant stress . The actively secreted form of HMGB1 is believed to be at least partially acetylated, although both actively and passively released HMGB1 will promote inflammation .
An early observation dating back to 1973 is that the HMG proteins aggregate with less basic proteins . HMGB1 binds LPS and a variety of cytokines such as IL-1β. This results in increased interferon gamma (INFγ) production by PBMC (peripheral blood mononuclear cells) that is much greater than with just HMGB1 or cytokines alone. HMGB1 binding to RAGE is enhanced with CpG DNA. HMGB1's ability to activate RAGE may result more from its ability to form a complex with other pro-inflammatory molecules, with this complex subsequently activating RAGE . Therefore any test of RAGE binding solely by HMGB1 will have to account for this, since contamination with even small amounts of LPS or CpG DNA will increase binding. Thrombomodulin competes with RAGE for HMGB1 in vitro and the resulting complex does not appear to bind RAGE, suggesting a possible approach to attenuate RAGE-HMGB1 signaling [78, 94]. In fact binding to thrombomodulin can also lead to proteolytic cleavage of HMGB1 by thrombin, resulting in a less-active inflammatory product .
A peptide consisting of only residues 150–183 of HMGB1 (the end of the B-box and its linker to the acidic tail) exhibits RAGE binding and successfully competes with HMGB1 binding in vitro . This sequence ias similar to the first 40 amino acids (the first EF-hand helix-loop-helix sequence) of several S100 proteins. An HMGB1 mutant in which amino acids 102–105 (FFLF, B-box middle) are replaced with two glycines induces significantly less TNFα release relative to full length HMGB1 in human monocyte cultures . This mutant is also able to competitively inhibit HMGB1 simulation in a dose-dependent manner when both are added.
Is HMGB1 the lone RAGE activator of the HMG family?
For all the reasons noted above, HMGB1 is the sole known HMG-box ligand of RAGE. None of the other nuclear HMG proteins have been shown to activate RAGE. The HMGB proteins can complex CpG DNA, and highly bent structures such as four-way Holliday junctions and platinated/platinum-modified DNA while other members cannot. Unlike other HMGB proteins, HMGB1 is abundantly expressed in nearly all tissues, and thus is readily available for translocation out of the nucleus to the cytosol for active and passive secretion. Although as a cautionary note, HMGB2 and HMGB3 are also upregulated in some cancers, and might play a role as RAGE activators in addition to HMGB1. The similarity of these proteins to HMGB1 suggests in various assays that they may be misidentified and included in the reported HMGB1 levels. The HMG and S100 family members each consist of similar proteins that have distinct and often unapparent RAGE-activating properties.
S100 Proteins as RAGE ligands and their role in Inflammation
A recent review on S100 proteins has been published, and provides more extensive detail than given here . We will focus on the critical elements necessary to consider their role in cancer and inflammation. S100 proteins are a family of over 20 proteins expressed in vertebrates exclusively and characterized by two calcium binding EF-hand motifs connected by a central hinge region . Over forty years ago the first members were purified from bovine brain and given the name "S-100" for their solubility in 100% ammonium sulfate . Many of the first identified S100 proteins were found to bind RAGE, and thus RAGE-binding was theorized to be a common property of all S100 proteins. However several of the more recently identified members of the family do not bind RAGE. The genes located on a cluster on human chromosome 1q21 are designated as the s100a sub-family and are numbered consecutively starting at s100a1. The S100 genes elsewhere are given a single letter, such as s100b . In general, mouse and human S100 cDNA is 79.6–95% homologous although the mouse genome lacks the gene for S100A12/EN-RAGE . Most S100 proteins exist as non-covalent homodimers within the cell . Some form heterodimers with other S100 proteins – for example the S100A8/S100A9 heterodimer is actually the preferred form found within the cell. The two EF-hand Ca++ binding loops are each flanked by α-helices. The N-terminal loop is non-canonical, and has a much lower affinity for calcium than the C-terminal loop. Members of this family differ from each other mainly in the length and sequence of their hinge regions and the C-terminal extension region after the binding loops. Ca++ binding induces a large conformational change which exposes a hydrophobic binding domain (except for S100A10 which is locked in this conformation) . This change in conformation allows an S100 dimer to bind two target proteins, and essentially form a bridge between as a heterotetramer . The S100 proteins have been called "calcium sensors" or "calcium-regulated switches" as a result. Some S100 proteins also bind Zn++ or Cu++ with high affinity, and this might affect their ability to bind Ca++ .
S100 proteins have wildly varying expression patterns (Table 3). They are upregulated in many cancers, although S100A2, S100A9, and S100A11 have been reported to be tumor repressors . S100 proteins and calgranulins are expressed in various cell types, including neutrophils, macrophages, lymphocytes, and dendritic cells . Phagocyte specific, leaderless S100 proteins are actively secreted via an alternative pathway, bypassing the Golgi . Several S100 proteins bind the tetramerization domain of p53, and some also bind the negative regulatory domain of p53. Binding of the tetramerization domain of p53 (thus controlling its oligomerization state) could be a property common to all S100 proteins but this has not been reported . Their roles in regulating the counterbalance between autophagy and apoptosis have also not been reported.
Individual S100 proteins are prevalent in a variety of inflammatory diseases, specifically S100A8/A9 (which possibly signals through RAGE in addition to other mechanisms), and S100A12 (which definitely signals through RAGE). These diseases include rheumatoid arthritis, juvenile idiopathic arthritis, systemic autoimmune disease and chronic inflammatory bowel disease. Blockade of the S100-RAGE interaction with soluble RAGE in mice reduced colonic inflammation in IL-10-deficient mice, inhibited arthritis development, and suppressed inflammatory cell infiltration [43, 33, 32, 105]. Some S100 proteins have concentration-dependant roles in wound healing, neurite outgrowth, and tissue remodeling.
There are several important questions that need to be addressed when examining proposed S100-RAGE interactions: Does this interaction occur in vivo in addition to in vitro? Could the observed effects be explained by a RAGE-independent mechanism (or even in addition to a non-RAGE mechanism)? Is this interaction dependant on the oligomeric state of the S100 protein? (S100 oligomeric state is itself dependant on the concentration of Ca++ and other metal ions as well as the redox environment). One area that has not received much attention is the possibility of S100 binding to a soluble RAGE in the cytosol or nucleus (as opposed to extracellular soluble RAGE).
S100 Proteins are not universal RAGE ligands
Several of the S100 family members are not RAGE ligands. Although there is no direct way to identify RAGE binding ability based on the amino acid sequences of the S100 proteins, conclusions can be drawn based on common biochemical properties of the known S100 non-ligands of RAGE: The first is that the non-ligands often exhibit strong binding to Zn++. The second is that their Ca++ binding is hindered or different in some ways from the S100 RAGE ligands. The third is that their oligomerization state is altered or non-existent.
Non-ligands of RAGE: S100A2, A3, A5, A10, A14, A16, G, Z
S100A2 is a homodimer that can form tetramers upon Zn++ binding, and this Zn++ binding inhibits its ability to bind Ca++. Although two RAGE ligands (S100B and S100A12) also bind Zn++ very well, the effect on them is to increase their affinity for Ca++ [106, 107]. The related S100A3 binds Ca++ poorly but Zn++ very strongly . S100A5 is also a Zn++ binder, but it binds Ca++ with 20–100 fold greater affinity than other S100 proteins. It also can bind Cu++, which will hinder its ability to bind Ca++ . S100A10 (or p11) is the only member of the S100 family that is Ca++ insensitive. It has amino acid alterations in the two Ca++ binding domains that lock the structure into an active state independently of calcium concentration . It will form a heterotetramer with Annexin A2, and it has been called "Annexin A2 light chain" . S100A14 has only 2 of the 6 conserved residues in the C-terminal EF-hand, and thus its ability to bind Ca++ is likely hindered . S100A16 binds Ca++ poorly, with only one atom per monomer of protein. However upon addition of Zn++, higher aggregates form . S100G was also known as Vitamin D-dependent calcium-binding protein, intestinal CABP, Calbindin-3, and Calbindin-D9k . It is primarily a monomer in solution and upon Ca++ binding it does not exhibit the conformational changes that characterize many other S100 proteins . S100Z is a 99-amino acid protein that binds S100P in vitro. It exists as a homodimer that binds Ca++ but its aggregation state is unaffected by Ca++ .
Possible ligands of RAGE: S100A1, S100A8/9
S100A1 normally exists as a homodimer, and its mRNA is observed most prominently in the heart, with decreasing levels in kidney, liver, skin, brain, lung, stomach, testis, muscle, small intestine, thymus and spleen. S100A1 is present in the cytoplasm and nucleus – rat heart muscle cell line H9c2 is mostly nuclear, adult skeletal muscle mostly cytoplasmic. S100A1 is released into the blood during ischemic periods, and extracellular S100A1 inhibits apoptosis via ERK1/2 activation . S100A1 binds to both the tetramerization and negative regulatory domains of p53 . S100A1 interacts with S100A4 and they antagonize each other in vitro and in vivo . There is still some debate if S100A1 binds to RAGE, although recent work with PET Imaging of Fluorine-18 labeled S100A1 administered to mice indicates that it co-localizes with RAGE .
In addition to forming homodimers, S100A8 and S100A9 can form heterodimers and heterotetramers with each other in a calcium and oxidation-dependant fashion . S100A8 and S100A9 have not been directly shown to activate RAGE, but there is substantial functional evidence that many of their effects are blocked by RAGE suppression or silencing. S100A8/9 exerts a pro-apoptotic effect in high concentrations, but promotes cell growth at low concentrations . The effects of N-carboxymethyl-lysine-modified S100A8/9 are ameliorated in RAGE knockout mice or by administration of soluble RAGE to wild-type mice . S100A8/9 binds to heparan sulfate, proteoglycans, and carboxylated N-glycans . A small (<2%) sub-population of RAGE expressed on colon tumor cells are modified to carboxylated N-glycans, and it is quite possible that this sub-population is activated by S100A8/9 . S100A8 and S100A9 proteins are secreted in an energy-dependent fashion by phagocytes during inflammatory processes. They stimulate specific inflammatory patterns in endothelial cells, interacting with components of the cytoskeleton, including keratin filaments, microtubules, type III intermediate filaments, and F-actin . In the presence of calcium, S100A8 and S100A9 form tetramers and bind directly to microtubules. S100A8/S100A9 also modulates tubulin-dependent cytoskeleton rearrangement during migration of phagocytes. S100A8 and S100A9 interact with both type III intermediate filaments and keratin filaments for the purpose of wound repair. Extracellularly, the S100A8/S100A9 complex displays cystostatic and antimicrobial activities and inhibits macrophage activation and immunoglobulin synthesis by lymphocytes . The reduced but not the oxidized S100A8 homodimer is strongly chemotactic for leukocytes . Upregulation of S100A8 and S100A9 in premetastatic lung tissue provide a niche for migration of tumor cells . This upregulation can be induced by VEGF-A, TGFβ and TNFα secretion from distant tumors. Upregulation in lung VE-cadherin+ endothelial cells promotes recruitment and infiltration of Mac1+ myeloid cells, and thus provides a niche for migration of tumor cells. Blocking S100A8 and S100A9 expression in the premetastatic stage could prevent this permissive niche from being formed and thus inhibit the migration of tumor cells.
Ligands of RAGE: S100A4, A6, A7/A7A/A15, A11, A12, A13, B, P
S100A4 binds to RAGE, and has been implicated in upregulation of MMP-13 (Matrix Metalloproteinase 13) in osteoarthritis, which leads to tissue remodeling . S100A4 is expressed in astrocytes, Schwann cells, and other neuronal cells in addition to chondrocytes . S100A4 is upregulated after nerve tissue injury. Neurite outgrowth stimulated by S100A4 is observed for the protein in the oligomeric, not dimeric, state . This protein also stimulates angiogenesis via the ERK1/2 signaling pathway. S100A4 binds to the tetramerization domain but not the negative regulatory domain of p53 .
S100A6 is found primarily in the neurons of restricted regions of the brain . S100A6 is also found in the extracellular medium of breast cancer cells. S100A6 binds to the tetramerization domain of p53 . S100A6 bound significantly to the C2 domain of RAGE, as opposed to the V and/or C1 domains to which most other ligands bind and thus suggests that it might have a discordant function from other RAGE ligands. S100A6 triggers the JNK pathway and subsequently the Caspase 3/7 pathway, resulting in apoptosis .
S100A7, also called Psoriasin 1, is part of a sub-family of several proteins . The highly homologous S100A7A, a member of this sub-family, was formerly known as S100A15 but this name has been withdrawn . S100A7 and S100A7A are functionally distinct despite the high sequence similarity . S100A7 is highly expressed in epidermal hyperproliferative disease and recruits CD4+ lymphocytes and neutrophils . Although several S100 proteins are upregulated in various forms of breast cancer, S100A7 is strongly up-regulated only in ductal carcinoma in situ . There are high levels of monomeric and covalently crosslinked high molecular weight S100A7 in human wound exudate and granulation tissue. Immunohistological studies suggest that S100A7 is produced by keratinocytes surrounding the wound and is released into the wound exudate. S100A7 exerts antibacterial activity, and the central region including only amino acids 35–80 is sufficient to mediate this activity . Although both S100A7 and S100A7A are expressed in keratinocytes, S100A7A is also expressed in melanocytes and Langerhans cells of the epidermis, and dermal smooth muscle endothelial cells . Binding, signaling, and chemotaxis of S100A7 are dependent on Zn++ and RAGE in vitro, while S100A7A seems to signal through a RAGE-independent pathway. S100A7 and S100A7A exert a synergistic effect, promoting inflammation in vivo .
S100A11 is overexpressed in many cancers . It is homodimeric and interacts in a Ca++ dependant fashion with annexin I . It is a key mediator of growth of human epidermal keratinocytes triggered by high Ca++ or TGFβ . Under these conditions S100A11 is phosphorylated and transported to the nucleus by nucleolin. S100A11 binds to the tetramerization domain (but not the negative regulatory domain) of p53 . Extracellular S100A11 is dimerized by transglutaminase 2, and this covalent homodimer acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couples inflammation with chondrocyte activation to promote osteoarthritis progression .
S100A12 (EN-RAGE) is primarily expressed in granulocytes, but also in found in keratinocytes and psoriatic lesions. S100A12 represents about 5% of the total cytosolic protein in resting neutrophils. It is expressed in acute, chronic, and allergic inflammation. It interacts with RAGE in a Ca++ dependent manner, but also binds Cu++. There is no s100a12 gene in mice, although S100A8 seems to be a functional homologue [132, 133]. S100A12 is up regulated in psoriasis and melanoma . It binds to the RAGE C1 and C2 domains instead of the V domain . It can also bind to RAGE expressed on endothelial cells, signaling through the NF-κB and MAPK pathways. S100A12 shares sequence homology with the putative RAGE-binding domain of HMGB1 (residues 153–180). Secreted S100A12 binds to RAGE and enhances expression of intercellular adhesion molecule-I (ICAM-1), vascular cell adhesion molecule-I (VCAM-1), NF-κB, and tumor necrosis factor (TNF)-α . S100A12 is a chemoattractant for monocytes and mast cells, although only the hinge region seems important for the latter . Since mast cells do not express RAGE protein or mRNA, their activation by S100A12 occurs in a RAGE-independent fashion. S100A12 exists as a homodimer under low Ca++ conditions, but will form hexamer aggregates (three dimers) at millimolar concentrations of Ca++ . S100A12, in addition to S100A13, binds to the anti-allergic drugs cromolyn, tranilast, and amlexanox in a Ca++ dependant manner. This suggests that S100A12 and S100A13 might be involved in degranulation of mast cells in a RAGE-independent manner .
S100A13 has a very broad expression pattern, in contrast to the other S100 proteins. S100A13 is expressed in endothelial cells, but not vascular smooth muscle cells. It is upregulated in extra-uterine endometriosis lesions when compared to normal tissues, and may have a role in vascularization . Its affinity for Ca++ is low, but Ca++ binding leads to a conformational change exposing a novel Cu++ binding site . Upon Cu++ binding, it regulates the stress-dependant release of FGF-1 and plays a role in angiogenesis in high-grade astrocytic gliomas . S100A13 in addition to S100A12 may be involved in the degranulation of mast cells .
S100B is expressed primarily in the astrocytes of the human cortex and melanocytes as well as myeloid dendritic cells . S100B, along with S100A1 and S100A6, are the most abundant S100 proteins in the brain of several species including mice and rats. Elevated levels of S100B have been found in patients following brain trauma, ischemia/infarction, Alzheimer's disease, and Down's syndrome . S100B is used as a marker of glial cell activation and death . It is believed to exist as a mixture of covalent and non-covalent dimers in the brain since ELISA assays done under non-oxidizing conditions will underestimate the amount of S100B [141, 142]. In this regard, covalent S100B dimers can be used as a marker of oxidative stress . S100B binds to both the tetramerization domain and the negative regulatory domain of p53 . S100B also inhibits microtubulin and type III intermediate filament assemblies. S100B binds both the variable (V) and constant (C1) regions of RAGE, and oligomers of S100B bind RAGE more strongly [42, 48]. At equivalent concentrations, S100B increases cell survival while S100A6 induces apoptosis via RAGE interactions, dependant on generation of reactive oxygen species (ROS). Upon binding to RAGE and activating intracellular ROS formation, S100B activates the PI 3-kinase/AKT pathway and subsequently the NFκB pathway, resulting in cellular proliferation. S100B exerts trophic effects on neurons and astrocytes at lower concentrations and causes neuronal apoptosis, activating astrocytes and microglia at higher concentrations [143–146]. S100B activation of RAGE upregulates IL-1β and TNF-α expression in microglia and stimulates AP-1 transcriptional activity through JNK signaling. Upregulation of COX-2, IL-1β and TNF-α expression in microglia by S100B requires the concurrent activation of NF-κB and AP-1.
S100P binds to RAGE and is important in prostate, pancreas, and gastric cancers [146, 147]. It is also detected in normal lung as well as lung cancer tissue, and is increased primarily in adenocarcinomas . Treatment of pancreatic cell lines with S100P stimulates cell proliferation, migration, invasion, and activates the MAP kinase and NFκB pathways . The anti-allergy drug cromolyn binds S100P and will block S100P-RAGE interaction. It inhibits tumor growth and increases the effectiveness of gemcitabine in experimental animal models . Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are simultaneously pro-tumorigenic by up-regulating S100P expression and anti-tumorigenic by decreasing Cox2 activity .
S100 Proteins – subtle differences translate to large changes in RAGE binding
Although the S100 proteins share much structural similarity with their two EF-hand Ca++ binding domains flanked by α-helices, only some of the members activate RAGE . Subtle structural differences that lead to different biochemical properties (Ca++ and Zn++ binding and preferred oligomerization state) thus seem to lead to different abilities to activate RAGE. Higher oligomerization states tend to lead to RAGE activation. RAGE also binds to several protein families that readily form aggregates and oliogmers – Amyloid beta peptide, Collagen, and AGEs.
RAGE and Abeta
The Amyloid-beta peptide (Abeta) is a peptide most commonly of 40 or 42 amino acids whose accumulation in amyloid plaques is one of the characteristics of Alzheimer brains. Abeta exists extracellularly either as a monomer, soluble oligomer, or insoluble fibrils and aggregates. Abeta binds to RAGE on neurons and microglial cells . On neurons, Abeta activation of RAGE will generate oxidative stress and activate NF-KB. Abeta activation of microglia will enhance cell proliferation and migration [153, 154]. However other receptors might also mediate Abeta toxicity, since RAGE-independent effects also exist . The V and C1 domains of RAGE bind to Abeta oligomers and aggregates (respectively), and blocking these will prevent Abeta-induced neurotoxicity . Exposure of a RAGE-expressing human neuroblastoma cell line (SHSY-5Y) to Abeta oligomers caused massive cell death, while exposure to Abeta fibrils and aggregates caused only minor cell death. Treatment with blocking antibodies specific to RAGE domains was able to protect against Abeta aggregate- or oligomer-inducuded death (but not fibril-induced death).
RAGE and Collagen
Unlike other non-embryonic tissues, RAGE is highly expressed in healthy lung and its expression decreases in pathological states. RAGE expression in the lung is a differentiation marker of alveolar epithelial type I (AT I) cells, and is localized to the basolateral plasma membrane . RAGE enhances adherence of these cells to collagen-coated surfaces and induces cell spreading . RAGE binds laminin and Collagen I and IV in vitro, but not fibronectin. Thus RAGE plays a role in anchoring AT I cells to the lung basement membrane, which is rich in Collagen IV [20, 157, 158]. Absence of RAGE expression in (-/-) mice leads to an increase in spontaneous idiopathic pulmonary fibrosis (IPF). Human lung from late-stage IPF patients showed significant down-regulation of RAGE when compared to healthy lung tissue .
Advanced glycation endproducts (AGEs) a broad class of non-enzymatic products of reactions between proteins or lipids and aldose sugars . The reaction between the protein and sugar causes its characteristic browning in food products. The western diet in particular is full of AGEs. Although glycation is a general term for addition of a sugar, in this case it specifically refers to non-enzymatic addition to a protein. "Glycosylation" is often used for enzymatic addition of sugars. The Maillard reaction, starting from the glycation of protein and progressing to the formation of AGEs, is implicated in the development of complications of diabetes mellitus, as well as in the pathogenesis of cardiovascular, renal, and neurodegenerative diseases [3, 119, 160, 161]. The Maillard reaction begins with the sugars forming Schiff bases and Amadori products. The carbonyl groups of these precursors can react with amino, sulfhydryl, and guanidinyl functional groups in proteins. AGEs cannot be chemically reverted to their original forms but their precursor, Amadori products, can be. AGEs are a diverse category of non-enzymatic modifications that result for these reactions, and not all AGE-modified proteins activate RAGE. Over twenty different AGE modifications have been characterized, of which carboxymethyl lysine (CML) modified proteins are strong inducers of RAGE signaling [3, 160]. Other AGE modifications to proteins (such as pentosidine and pyrraline) do not increase RAGE signaling. As such, characterizing AGE-modifications of proteins is important. One promising technique is Mass Spectrometry, especially "bottom-up" proteomics involving cleavage of proteins followed by analysis of the subsequent peptides .
RAGE and AGEs in the Redox Environment
AGE accumulation itself is considered a source of oxidative stress. In hyperglycemic environments, glucose can undergo auto-oxidation and generate OH radicals [161, 162]. Schiff-base products and Amadori products themselves cause ROS production . Nitric Oxide donors can scavenge free radicals and inhibit AGE formation . Over time AGE deposits contribute to diabetic atherosclerosis in blood vessels. As a human naturally ages, one generates high levels of endogenous AGEs [164, 165].
RAGE was originally named for its ability to bind AGEs, but since 1995 there have been many more ligands found [8, 166]. Formation of AGEs is a way to sustain the signal of a short oxidative burst into a much longer-lived post-translationally modified protein . RAGE will bind to AGE-modified albumin but not nonglycated albumin . AGE activation of RAGE is found in diabetes, neuodegeneration, and aging . Tumors provide an environment that favors generation of AGEs since according to Warburg's original hypothesis they rely primarily on anaerobic glycolosis for energy, and have a higher uptake of glucose [169, 170]. Prostate carcinoma cells bind AGEs through the V-domain of RAGE . AGEs have in fact been identified in cancerous tissue, which leads to the possibility of AGE activation of RAGE contributing to cancer growth . However there are also other RAGE ligands in greater abundance. Single molecules of RAGE do not bind AGEs well, but oligomers of RAGE bind them strongly . This supports the notion that RAGE oligomerization is important for sustained signaling. Collagen will normally accumulate some degree of glycation in vivo, but collagen with synthetic AGE-modification will enhance neutrophil adhesion and spreading .
Sorbinil and zenarestat are orally active aldose reductase inhibitors (ARI's) derived from quinazoline. They, in addition to vitamin C and E, have ameliorative benefits in decreasing intracellular oxidative stress . Vitamin E is effective in part because of its chemical structure. It is able to donate a hydrogen atom from its hydroxyl group, combining with ROS and neutralizing them . Sadly, many of the clinical trials of antioxidants have failed to modify cancer and have in some instances enhanced its development, suggesting that "aerobic" or oxidative extracellular events may be a preferred means to limit chronic inflammation. Injection of soluble RAGE prevents liver reperfusion injury and decreases levels of TNF-α (Tumor Necrosis Factor-α), a cytokine that signals apoptosis and contributes to systemic inflammation, and thereby decreases insulitis . Aminoguanidine delivery also decreases levels of albumin in the blood stream and decreases aortic and serum levels of AGEs thus slowing the progression of atherosclerosis .
RAGE Ligands in Neurobiology
The RAGE-NF-κB axis operates in diabetic neuropathy. This activation was blunted in RAGE (-/-) mice, even 6 months following diabetic induction. Loss of pain perception is reversed in wild type mice treated with exogenous soluble RAGE . The interaction between HMGB1 and RAGE in vitro promotes neurite outgrowth of cortical cells, suggesting a potential role of RAGE as a mediator in neuronal development . Nanomolar concentrations of S100B promote cell survival responses such as cell migration and neurite growth. While the interaction of RAGE with S100B can produce anti-apoptotic signals, micro-molar concentrations of S100B will produce oxyradicals, inducing apoptosis. S100B also activates RAGE together with HMGB1, promoting the production of the transcription factor NF-kB . Another proposed mechanism for how RAGE may mediate neurite outgrowth involves sulfoglucuronyl carbohydrate (SGC). Examination of both HMGB1 and SGC in the developing mouse brain reveals that the amount of RAGE expressed in the cerebellum increases with age. Antibodies to HMGB1, RAGE, and SGC inhibit neurite outgrowth, suggesting that RAGE may be involved with the binding of these molecules and their downstream processes .
As RAGE may be involved with cell growth and death, its role in cell recovery after injury has also been examined. In rats with permanent middle cerebral artery occlusion, levels of RAGE increase as they do in PC12 cells following oxygen and glucose deprivation (OGD). Blockade of RAGE reduces cytotoxicity caused by OGD . Binding of RAGE to its ligands activates the NF-κB pathway. The presence of RAGE, NF-κB, and NF-κB regulated cytokines in CD4+, CD8+, and CD68+ cells recruited to nerves of patients with vasculitic neuropathies suggests that the RAGE pathway may also play a role in the upregulation of inflammation in this setting . Another RAGE ligand, AGE-CML, is present in endoneurial and epineurial mononuclear cells in chronic inflammatory demyelinating polyneuropathy and vasculitic polyneuropathy .
In glioma cells, RAGE is part of a molecular checkpoint that regulates cell invasiveness, growth, and movement. In contrast to lung cancer cells, normal glioma cells express less RAGE than tumor cells. Addition of AGEs to cells stimulates proliferation, growth, and migration. Addition of antibodies targeting RAGE conversely inhibits the growth and proliferation caused by AGEs, increasing survival time and decreasing metastases in immunocompromised mice bearing implanted rat C6 glioma cells .
RAGE in Epithelial Malignancies
The interaction between RAGE and its various ligands plays a considerable role in the development and metastasis of cancer. RAGE impairs the proliferative stimulus of pulmonary and esophageal cancer cells . RAGE is highly expressed in Type-I pneumatocytes, specifically localized in the alveolar epithelium. Interestingly, over-expression of RAGE leads to lower cell proliferation and growth, while downregulation of RAGE promotes development of advanced stage lung tumors [19, 185]. Furthermore, blocking AGE-RAGE interactions leads to diminished cell growth . Cells expressing RAGE have diminished activation of the p42/p44-MAPK pathway and growth factor production (including IGF-1) is impaired. RAGE ligands detected in lung tumors include HMGB1, S100A1, and S100P. In pulmonary cancer cells transfected with a signal-deficient form of RAGE lacking the cytoplasmic domain, increased growth when compared to fl-RAGE-transfected cells is noted. Over-expression of RAGE on pulmonary cancer cells does not increase cell migration, while signal deficient RAGE does .
RAGE and Immune Cells
RAGE also acts as an endothelial adhesion receptor that mediates interactions with the β2 integrin Mac-1 . HMGB1 enhances RAGE-Mac1 interactions on inflammatory cells, linking it to inflammatory responses (Table 4) [71, 72]. Neutrophils and myelomonocytic cells adhere to immobilized RAGE or RAGE-transfected cells, and this interaction is attributed to Mac-1 interactions [24, 71]. RAGE is highly expressed in macrophages, T lymphocytes, and B lymphocytes . RAGE expressed on these cell types contributes to inflammatory mechanisms. The activation of RAGE on T-Cells is one of the early events that leads to the differentiation of Th1+ T-Cells . RAGE is also a counter-receptor for leukocyte integrins, directly contributing to the recruitment of inflammatory cells in vivo and in vitro. Soluble RAGE has been postulated as a direct inhibitor of leukocyte recruitment . RAGE-mediated leukocyte recruitment may be particularly important in conditions associated with higher RAGE expression, such as diabetes mellitus, chronic inflammation, atherosclerosis or cancer . RAGE can directly mediate leukocyte recruitment, acting as an endothelial cell adhesive receptor and attracting leukocytes. RAGE causes an "indirect" increase in inflammatory cell recruitment due to RAGE-mediated cellular activation and upregulation of adhesion molecules and proinflammatory factors . S100A12 and S100B activate endothelial, vascular smooth muscle cells, monocytes and T cells via RAGE, resulting in the generation of cytokines and proinflammatory adhesion molecules [24, 67, 68].
RAGE expression on T cells is required for antigen-activated proliferative responses . RAGE deficient T cells decrease production of IL-2, IFN-γ, and Th1 while producing more IL-4 and IL-5 as Th2 cytokines. RAGE activation thus plays a role in balancing Th1 and Th2 immunity. RAGE deficient dendritic cells appear to mediate rather normal antigen presentation activity and migration both in vivo and in vitro. RAGE expression is however required by maturing DCs to migrate to draining lymph nodes .
Crucial role of RAGE in inappropriate increase of smooth muscle cells from patients with pulmonary arterial hypertension
Background: Pulmonary vascular remodeling of pulmonary arterial hypertension (PAH) is characterized by an inappropriate increase of vascular cells. The receptor for advanced glycation end products (RAGE) is a type I single-pass transmembrane protein belonging to the immunoglobulin superfamily and is involved in a broad range of hyperproliferative diseases. RAGE is also implicated in the etiology of PAH and is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with PAH. We examined the role of RAGE in the inappropriate increase of PASMCs in patients with PAH.
Methods and results: PASMCs were obtained from 12 patients with PAH including 9 patients with idiopathic PAH (IPAH) and 3 patients with heritable PAH (HPAH) (2 patients with BMPR2 mutation and one patient with SMAD9 mutation) who underwent lung transplantation. Western blot analysis and immunofluorescence staining revealed that RAGE and S100A8 and A9, ligands of RAGE, were overexpressed in IPAH and HPAH-PASMCs in the absence of any external growth stimulus. PDGF-BB (10 ng/mL) up-regulated the expression of RAGE in IPAH and HPAH-PASMCs. PAH-PASMCs are hyperplastic in the absence of any external growth stimulus as assessed by 3H-thymidine incorporation. This result indicates overgrowth characterized by continued growth under a condition of no growth stimulation in PAH-PASMCs. PDGF-BB stimulation caused a higher growth rate of PAH-PASMCs than that of non-PAH-PASMCs. AS-1, an inhibitor of TIR domain-mediated RAGE signaling, significantly inhibited overgrowth characterized by continued growth under a condition of no growth stimulation in IPAH and HPAH-PASMCs (P<0.0001). Furthermore, AS-1 significantly inhibited PDGF-stimulated proliferation of IPAH and HPAH-PASMCs (P<0.0001).
Conclusions: RAGE plays a crucial role in the inappropriate increase of PAH-PASMCs. Inhibition of RAGE signaling may be a new therapeutic strategy for PAH.
Conflict of interest statement
The authors have declared that no competing interests exist.
Fig 1. RAGE expression in PASMCs of…
Fig 1. RAGE expression in PASMCs of patients with PAH.
A. Immunohistochemical staining of RAGE…
Fig 2. S100A8/A9 expression in PASMCs of…
Fig 2. S100A8/A9 expression in PASMCs of patients with PAH.
A. Immunohistochemical staining of S100A8…
Fig 3. Increased expression of RAGE in…
Fig 3. Increased expression of RAGE in PASMCs of patients with PAH by PDGF-BB stimulation…
Fig 4. Inhibitory effects of AS-1 or…
Fig 4. Inhibitory effects of AS-1 or RAGE aptamer on proliferation of PAH-PASMCs assessed by…
Inflammation plays a key role in atherosclerosis. We hypothesized that novel inflammatory markers may predict the risk of coronary heart disease (CHD).
Approach and Results—
We investigated the association of 16 inflammatory biomarkers with the risk of CHD in a random subset of 839 CHD-free individuals in a prospective population-based cohort study. A Bonferroni corrected P value of 3.1×10 −3 was used as a threshold of statistical significance. The mean age at baseline was 72.8 years. During a median follow-up of 10.6 years, 99 cases of incident CHD were observed. Among all inflammatory biomarkers, neutrophil-derived human s100a12 (extracellular newly identified receptor for advanced glycation end-products binding protein [EN-RAGE]) showed the strongest association with the risk of CHD (P value 2.0×10 −3 ). After multivariable adjustment for established cardiovascular risk factors, each standard deviation increase in the natural log-transformed EN-RAGE was associated with 30% higher risk of incident CHD (hazard ratio, 1.30 95% confidence interval, 1.06–1.59). Further adjustment for previously studied inflammatory markers did not attenuate the association. Excluding individuals with prevalent type 2 diabetes mellitus, impaired kidney function, or individuals using antihypertensive medication did not change the effect estimates. Cause-specific hazard ratios suggested a stronger association between EN-RAGE and CHD mortality compared with stable CHD.
Our results highlight EN-RAGE as an inflammatory marker for future CHD in a general population, beyond traditional CHD risk factors and inflammatory markers.
With 7.3 million deaths per year globally, coronary heart disease (CHD) is still the world’s leading cause of mortality. 1 Inflammation is thought to play a key role in the pathogenesis of atherosclerosis and CHD. 2 Accordingly, inflammatory markers have been investigated for predicting the risk of CHD, an effort that has led to the identification and validation of several inflammatory markers for CHD. 3–6 However, the inflammatory markers that have been investigated to date only represent a minor part of the diverse molecules that constitute the complex human immune response. 7 Exploring prospectively the association of relatively uninvestigated inflammatory markers with CHD may unravel novel inflammatory risk factors for CHD and may shed light on additional pathways that might be involved in the pathogenesis of atherosclerosis and CHD.
We hypothesized that indicators of inflammation which have not been studied previously with the incidence of CHD are associated with incident CHD beyond traditional risk factors and previously studied inflammatory markers. To this end, we studied the association of 16 biomarkers of inflammation with the risk of CHD in the Rotterdam Study, a prospective population-based cohort study.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Table 1 summarizes the baseline characteristics of 839 participants (see Table II in the online-only Data Supplement for baseline characteristics in future CHD cases and noncases). At the start of the study, the mean (±SD) age was 72.8 (7.5), and 58% of the population were female. During a median follow-up of 10.6 years (interquartile range, 6.8–11.9), 2 were lost to follow-up, 353 individuals died (302 unrelated to CHD), and 99 developed CHD (incidence rate, 12.7 per 1000 person years). Out of the 16 inflammatory biomarkers (Figure 1), after Bonferroni correction, only extracellular newly identified receptor for advanced glycation end-products binding protein (EN-RAGE) was significantly associated with CHD when adjusted for age and sex (Table III in the online-only Data Supplement). The risk of CHD was nearly one third increased per standard deviation increase in the natural log-transformed EN-RAGE (hazard ratio [HR], 1.37 95% confidence interval (CI), 1.12–1.67 Table 2). Compared with the lowest tertile, participants in the highest tertile experienced approximately a 2.6-fold higher risk of developing CHD compared with participants in the lowest tertile (HR, 2.59 95% CI, 1.52–4.40). When we further adjusted the association for traditional cardiovascular risk factors, the effect estimates attenuated slightly (HR, 1.30 95% CI, 1.06–1.59). Additional adjustment for previously studied inflammatory markers yielded slightly increased effect estimates (Table 2 Table IV in the online-only Data Supplement).
Figure 1. Flow chart of inflammatory biomarker inclusion.
Table 1. Baseline Characteristics of Participants at Risk for CHD
Plus–minus values are means±SD.
CHD indicates coronary heart disease.
* Values are presented as median (interquartile range).
Table 2. The Association Between EN-RAGE Serum Levels and Incident CHD
Model 1, adjusted for age and sex Model 2, adjusted for age, sex, BMI, systolic blood pressure, antihypertensive medication use, HDL cholesterol, total cholesterol, smoking status (current, noncurrent), prevalent type 2 diabetes mellitus, and eGFR Model 3, additionally adjusted for CD40ligand, Complement 3, C-reactive protein, interleukin 8, interleukin 18, monocyte chemotactic protein 1, macrophage migration inhibitory factor, RANTES, Resistin, TNF receptor 2, and white blood cells.
BMI indicates body mass index CHD, coronary heart disease CI, confidence interval eGFR, estimated glomerular filtration rate EN-RAGE, extracellular newly identified receptor for advanced glycation end-products binding protein HDL, high-density lipoprotein HR, hazard ratio SD, standard deviation and TNF, tumor necrosis factor.
* Hazard ratios are represented per standard deviation increase in log-transformed EN-RAGE.
Cumulative incidence curves for the tertiles of EN-RAGE adjusted for competing risks are depicted in Figure 2. The 10-year probability of first incident event of CHD was 0.05 (95% CI, 0.03–0.08) for the first tertile, 0.11 (95% CI, 0.07–0.14) for the second tertile, and 0.14 (95% CI, 0.10–0.18) for the third tertile.
Figure 2. Cumulative incidence curves for first, second, and third tertile of serum EN-RAGE in relation to incidence of coronary artery disease adjusting for competing noncoronary heart disease death ≤10 years of follow-up. EN-RAGE indicates extracellular newly identified receptor for advanced glycation end-products binding protein.
After excluding participants with chronic kidney disease at baseline, the association between EN-RAGE and incident CHD attenuated slightly (1.28 95% CI, 1.03–1.59 Table 3). Excluding participants with type 2 diabetes mellitus, the effect estimates of the association between EN-RAGE and CHD did not change: hazard ratio 1.29 (95% CI, 1.04–1.60) in the fully adjusted model. Finally, after excluding participants taking antihypertensive medication, the hazard ratio did not change (HR, 1.40 95% CI, 1.05–1.87).
Table 3. The Association of EN-RAGE With CHD in Absence of Patients With CKD, T2D, or Antihypertensive Use
Model 1, adjusted for age and sex Model 2, adjusted for age, sex, BMI, systolic blood pressure, antihypertensive medication use, HDL cholesterol, total cholesterol, smoking status (current and noncurrent), prevalent type 2 diabetes mellitus, and eGFR.
BMI indicates body mass index CHD, coronary heart disease CI, confidence interval CKD, chronic kidney disease eGFR, estimated glomerular filtration rate EN-RAGE, extracellular newly identified receptor for advanced glycation end-products binding protein HDL, high-density lipoprotein HR, hazard ratio and T2D, type 2 diabetes mellitus.
* Hazard ratios are represented per standard deviation increase in log-transformed EN-RAGE.
Table 4 depicts the results for the associations between EN-RAGE and the different CHD manifestations separately. We observed the strongest association with CHD mortality (HR, 1.56 95% CI, 1.19–2.04) compared with myocardial infarction and revascularization, which were not significant. Further adjustment for the traditional cardiovascular risk factors did not change the effect estimate for CHD mortality. Cause-specific HRs were not significantly lower for revascularization compared with myocardial infarction (Lunn and McNeil, P=0.700), but they were borderline-significant for CHD mortality compared with revascularization (Lunn and McNeil, P=0.055).
Table 4. The Association Between EN-RAGE and Incident Myocardial Infarction, Coronary Revascularization, and CHD Mortality
Model 1, adjusted for age and sex Model 2, adjusted for age, sex, BMI, systolic blood pressure, antihypertensive medication use, HDL cholesterol, total cholesterol, smoking status (current and noncurrent), prevalent type 2 diabetes, and eGFR.
BMI indicates body mass index CHD, coronary heart disease CI, confidence interval eGFR, estimated glomerular filtration rate EN-RAGE, extracellular newly identified receptor for advanced glycation end-products binding protein HDL, high-density lipoprotein and HR, hazard ratio.
* Hazard ratios are represented per standard deviation increase in log-transformed EN-RAGE.
The c-statistic of the traditional Framingham risk score model for 10-year CHD risk was 0.730 (95% CI, 0.672–0.788). When we added EN-RAGE to the model, the c-statistic improved to 0.741 (95% CI, 0.683–0.799) with a difference of 0.011 (95% CI, −0.012 to 0.033 P=0.33). Adding EN-RAGE to the Framingham risk score model resulted in a significant continuous net reclassification index of 0.36 (95% CI, 0.05–0.67 P=0.02) and integrated discrimination improvement of 0.026 (95% CI, 0.009–0.0437 P=3.3×10 −03 ).
In this prospective, population-based cohort study, we found that higher EN-RAGE levels were associated with an increased risk of CHD beyond conventional risk factors. Further adjustments for inflammatory markers, as well as excluding diseased individuals, did not change the results. These findings suggest proinflammatory EN-RAGE as a new inflammatory risk marker for CHD that represents a distinct inflammatory pathway compared with other inflammatory markers.
Previous studies have observed increased levels of EN-RAGE in patients with chronic inflammatory disorders, including inflammatory bowel disease, 8 type 2 diabetes mellitus, 9 chronic kidney disease, subclinical atherosclerosis, 10,11 and coronary artery disease. 12–16 The design of the mentioned studies is mainly cross-sectional and conducted in patient populations. A positive association between EN-RAGE and CHD mortality was shown in a prospective study, including patients on hemodialysis. 17 In addition, a recent study in Japanese CHD individuals observed a significant association between EN-RAGE and future major adverse cardiac events. 18 To our knowledge, we are the first to investigate the association between EN-RAGE and CHD in a prospective population-based cohort study with long-term follow-up.
To address the possibility of confounding, we adjusted in the multivariable model for the different traditional CHD risk factors and previously studied inflammatory markers. To address the question whether our results were driven by a certain subgroup, we analyzed the data excluding participants with chronic kidney disease, type 2 diabetes mellitus, and antihypertensive use in the sensitivity analyses. Across all these analyses, there was a consistent effect of EN-RAGE on the risk of CHD, even after adjusting for the established inflammatory markers. These results suggest an effect of EN-RAGE on the risk of CHD beyond well-established metabolic and inflammatory pathways.
We observed a stronger association between EN-RAGE and future myocardial infarction and CHD mortality compared with revascularization. This suggests that EN-RAGE is more a determinant of acute coronary events with plaque instability rather than stable coronary artery disease. This observation that EN-RAGE, a member of the S100 protein family, is a strong determinant of acute coronary events is in line with previous studies that reported higher levels of mRNA and plasma levels of family S100 proteins (S100A8/9) in patients with ST-elevated myocardial infarction compared with stable coronary artery disease cases. 19 Furthermore, a postmortem study in people died from sudden cardiac death has found high expression levels of S100A12 in coronary artery smooth muscle in the ruptured plaques, especially in diabetics. 20 However, the cause-specific hazard ratio for the CHD events were not significantly different using the method proposed by Lunn and McNeil. We might have been underpowered to observe a significant difference because of the limited number of cases in this cause-specific analyses.
Studying the added value of EN-RAGE in 10-year CHD risk prediction, we found an improvement in risk prediction when we added EN-RAGE to the Framingham risk score. This suggests that EN-RAGE, as a noninvasive marker of future CHD, could be useful in predicting the risk of CHD in the general population. Although we corrected the change in c-statistic for optimism, we think that further studies are needed to establish the potential role of EN-RAGE in CHD risk prediction.
EN-RAGE, a member of the S100 protein family of EF-hand calcium-binding proteins, is an endogenously produced inflammatory ligand of the RAGE 21 and Toll-like receptor 4. 22 RAGE is a member of the immunoglobulin superfamily of cell surface molecules and is expressed in multiple tissues, including endothelium cells, vascular smooth muscle cells, and monocyte-derived macrophages. 23 The binding of RAGE by EN-RAGE activates inflammatory cascades, including the proinflammatory NF-κB signaling pathway, a well-known pathway of the innate immune system involved in the pathogenesis of CHD. 21,24 Moreover, intracellular signaling pathways triggered by EN-RAGE may alter gene expression and upregulate the synthesis of vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 synthesis. 21 Considering atherosclerosis as a chronic inflammatory disease, the engagement of RAGE by EN-RAGE may play an important role in the pathogenesis of atherosclerosis and subsequently CHD. In line with this evidence, the expression of EN-RAGE in vascular smooth muscle cells can modulate the remodeling of the aortic wall and stimulates cytokine production and increases oxidative stress. 25 Moreover, EN-RAGE accelerates atherosclerosis and vascular calcification in apolipoprotein E-null mice. 26 Recently, EN-RAGE has been shown to accelerate the development of cardiac hypertrophy and diastolic dysfunction in mice with chronic kidney disease. 27 The monocyte activation effect of EN-RAGE has also been observed to be Toll-like receptor 4–dependent. 22 It was demonstrated that EN-RAGE facilitates inflammatory monocyte activation by Toll-like receptor 4 and that this effect was modulated by RAGE. Toll-like receptors have been investigated extensively in the field of cardiovascular diseases because they are expressed in vascular and myocardial cell membranes. 28 The important role of EN-RAGE in the pathogenesis of atherosclerosis is further emphasized by a recent study where pharmacological inhibition of S100A12-mediated atherosclerosis improved atherosclerotic plaque features, including smaller necrotic cores, diminished calcification, and reduced number of inflammatory cells. 29
This study has certain strengths and limitations. The prospective population-based study design, the diversity of the available inflammatory biomarkers, and the long-term follow-up of CHD can be marked as the main strengths of the present study. In addition, our findings are robust regarding the strict Bonferroni P value we used as the threshold for significant associations in the first step. Several limitations should also be acknowledged. First, although we adjusted our analysis for different potential confounders, we cannot exclude the effect of unknown or unmeasured confounders. However, because we adjusted for the traditional and commonly used risk factors for CHD and inflammatory pathways, we think that EN-RAGE as a novel inflammatory marker for CHD is interesting because it might reflect other pathways that lead to CHD. Second, we had to exclude inflammatory biomarkers with low serum concentrations. Nonetheless, the selected biomarkers have >60% completeness of measurements, indicating acceptable quality of quantification. Third, our population is ≥55 years. Therefore, generalization of the results to a younger age category should be with caution. Our study only indicates an association we think that further studies are needed to establish the causal role of EN-RAGE in the pathogenesis of CHD.
In conclusion, our study suggests that higher levels of serum EN-RAGE are associated with incidence of CHD beyond conventional cardiovascular risk factors and inflammatory markers. These results provide evidence for a role of EN-RAGE in the development of CHD and suggest this marker as a potential target for drug therapy and risk prediction.
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Targeting RAGE Signaling in Inflammatory Disease
The receptor for advanced glycation end-products (RAGE) is a multiligand pattern recognition receptor implicated in diverse chronic inflammatory states. RAGE binds and mediates the cellular response to a range of damage-associated molecular pattern molecules (DAMPs) including AGEs, HMGB1, S100s, and DNA. RAGE can also act as an innate immune sensor of microbial pathogen-associated molecular pattern molecules (PAMPs) including bacterial endotoxin, respiratory viruses, and microbial DNA. RAGE is expressed at low levels under normal physiology, but it is highly upregulated under chronic inflammation because of the accumulation of various RAGE ligands. Blocking RAGE signaling in cell and animal models has revealed that targeting RAGE impairs inflammation and progression of diabetic vascular complications, cardiovascular disease (CVD), and cancer progression and metastasis. The clinical relevance of RAGE in inflammatory disease is being demonstrated in emerging clinical trials of novel small-molecule RAGE inhibitors.
Receptor for AGE (RAGE) is a multi-ligand member of the immunoglobulin superfamily of cell surface molecules. Engagement of RAGE by its signal transduction ligands evokes inflammatory cell infiltration and activation in the vessel wall. In diabetes, when fueled by oxidant stress, hyperglycemia, and superimposed stresses such as hyperlipidemia or acute balloon/endothelial denuding arterial injury, the ligand–RAGE axis amplifies vascular stress and accelerates atherosclerosis and neointimal expansion. In this brief synopsis, we review the use of rodent models to test these concepts. Taken together, our findings support the premise that RAGE is an amplification step in vascular inflammation and acceleration of atherosclerosis. Future studies must rigorously test the potential impact of RAGE blockade in human subjects such trials are on the horizon.
Ligand engagement of the receptor for advanced glycation end products (RAGE) evokes vascular inflammation and perturbation. In diabetes, when fueled by oxidation, hyperglycemia, and superimposed stresses, the ligand–RAGE axis accelerates atherosclerosis and neointimal expansion. Studies in rodent models of diabetes suggest that testing the impact of RAGE blockade in human subjects is logical.
The receptor for advanced glycation end products (RAGE) is a multi-ligand member of the immunoglobulin superfamily of cell surface molecules. 1,2 Its ability to recognize multiple classes of ligands, such as advanced glycation end products (AGEs), S100/calgranulins, amphoterin, amyloid-β peptide and β-sheet fibrils, and MAC-1, 3–8 suggests that the repertoire of RAGE-dependent effects in the tissues may be diverse. In this context, the function of this receptor does not appear to be to degrade/detoxify ligand, but rather, by RAGE cytosolic domain-triggered signal transduction, to propagate immune/inflammatory responses. 3,9
Although AGEs are an heterogeneous class of compounds, studies have shown that a particular class of these species, N ε carboxymethyl lysine (CML) adducts of proteins/lipids, are specific signal transduction ligands of RAGE. 9 CML-modified adducts may be generated by a variety of stimuli. In addition to generation in hyperglycemia, CML adducts may also be generated by activation of the myeloperoxidase pathway. 10 Recent studies have shown that peroxynitrite may induce formation of CML by cleavage of an Amadori product and, also, by the generation of reactive α-oxoaldehydes from glucose. 11 These considerations highlight the concept that once set in motion, diabetes-associated hyperglycemia and oxidant stress magnify production of a wide array of biochemical species that accumulate to stimulate cellular activation and sustain tissue perturbation.
Furthermore, in diabetes, distinct mechanisms may be engaged to further fuel the cycle of AGE generation and oxidant stress. 12 One specific example is the polyol pathway. Glucose is reduced to sorbitol by aldose reductase, the central enzyme of this pathway. This results in generation of fructose fructose is converted into fructose-3-phosphate by the action of 3-phosphokinase. A chief product of this reaction is 3-deoxyglucosone, a key precursor in the generation of multiple types of AGEs, such as CML adducts and others. 13,14 It has been shown that in renal failure, plasma levels of 3-deoxyglucosone increase in parallel with increased levels of aldose reductase in erythrocytes. 14 Administration of an inhibitor of aldose reductase, epalrestat, to subjects with diabetes resulted in reduced levels of CML adducts and their precursors in erythrocytes and decreased plasma levels of thiobarbituric acid reactive substances. 15 These considerations highlight the concept that multiple forces are in play in diabetes to augment generation of tissue injurious biochemical species that signal chronic cellular perturbation.
Indeed, once AGEs are formed, their interaction with RAGE triggers generation of proinflammatory cytokines, adhesion molecules, and chemokines. Attraction of inflammatory cells such as polymorphonuclear leukocytes, mononuclear phagocytes, and T-lymphocytes into the tissue provides a mechanism to sustain the inflammatory response. In the context of RAGE, these cells may harbor proinflammatory S100/calgranulins and amphoterin. Once these molecules are released into the tissue, S100/calgranulins/amphoterin–RAGE interaction may amplify tissue inflammation and injury by autocrine and paracrine pathways. 3,16–18
Models of Atherosclerosis in Diabetes
Multiple epidemiologic investigations support the premise that diabetes accelerates atherosclerosis human subjects with diabetes display a striking acceleration of atherosclerosis, in addition to the increased incidence of cardiac events, such as heart attacks and strokes. 19–22 Studies in human subjects indicate that RAGE and its signal transduction ligands are expressed in diabetic vasculature and atherosclerotic plaques. 23,24 Cipollone et al showed that compared with nondiabetic human plaques, diabetic plaques were characterized by greater numbers of mononuclear phagocytes, T-lymphocytes, and HLA-DR + cells, in parallel with increased expression of RAGE, activated NF-kB, cox-2, and matrix metalloproteinases. The extent of RAGE expression was found to correlate with levels of glycosylated hemoglobin. 24 Although such studies do not delineate the biochemical/molecular link between the ligand/RAGE axis, inflammation, and atherosclerosis, these experiments nevertheless provide support for the premise that RAGE may participate in augmentation of vascular injury and progression of atherosclerosis.
Studies in murine models are underway to test the premise that RAGE contributes to the acceleration of atherosclerosis in diabetes.
Animal Models and Accelerated Diabetic Atherosclerosis
The first murine model to support the concept that hyperglycemia accelerated atherosclerosis tested the hypothesis that induction of diabetes using multiple low-dose streptozotocin would result in enhanced atherosclerosis in mice fed Western-type diet. 25 In these first studies, wild-type BALB/c and C57BL/6 mice were used. In these animals, genetic manipulation was not used as a means to trigger/enhance the development of atherosclerosis. Rather, dietary modification induced hyperlipidemia. In this study, Kunjathoor et al showed that BALB/c mice fed an atherogenic diet and rendered diabetic with streptozotocin displayed a 17-fold increase in atherosclerotic lesion area compared with citrate-treated (nondiabetic) mice fed the same atherogenic diet. Although the lesions were limited to fatty streaks, histological studies nevertheless revealed the presence of both excess mononuclear phagocytes and smooth muscle cells in the plaques. 25 Interestingly, these investigators found that there were no differences in mean atherosclerotic lesion area between diabetic (streptozotocin) versus nondiabetic atherogenic diet-fed C57BL/6 mice. 25 These considerations underscore the critical influence that multiple factors may exert on atherosclerosis initiation and progress, such as genetic background, diet, and environment, especially in the setting of superimposed hyperglycemia.
In addition to dietary modulation of lipid profile, genetic modification provides a means to alter lipid profile in mice. To test the role of hyperglycemia in potentially accelerating atherosclerosis, we used mice in which genetic manipulation rendered the animals hyperlipidemic by deletion of apolipoprotein E (apo E−/−). 26–27 Apo E−/− mice display lesions of multiple phases of atherosclerosis distributed throughout the arterial tree. 28 On normal chow diet, work from the Russell Ross group showed that apo E−/− mice displayed foam cell lesions as early as 8 weeks of age by age 15 weeks, advanced lesions were evident. 28 Advanced lesions consisted of a fibrous cap containing smooth muscle cells surrounded by a connective tissue matrix covering a necrotic core with foamy macrophages. 28 On induction of a Western-type diet, lesions generally were more advanced. 28
Based on the observation that apo E−/− mice developed complex lesions characteristic of advanced human plaques, we sought to test the hypothesis that induction of hyperglycemia in these animals would further accelerate atherosclerosis and lesion complexity. In the first studies, we rendered 6-week-old male apo E−/− mice diabetic with streptozotocin. 29 These apo E−/− mice were previously backcrossed >10 generations into C57BL/6. After 6 weeks of established diabetes, at age ≈13 to 14 weeks, diabetic apoE−/− mice exhibited discrete lesions at major branch points in the thoracic aorta and at the arch of the aorta. In contrast, age-matched euglycemic apoE−/− mice did not display lesions in the proximal aorta. 30 Quantitative morphometric analysis revealed ≈5-fold increase in mean lesion area at the level of the aortic sinus in diabetic apoE−/− mice compared with nondiabetic apoE−/− animals (Figure 1a). Histologic analysis of euglycemic apoE−/− mice revealed typical fatty streaks visualized with oil red O. At this age, the majority of lesions in the nondiabetic animals did not progress to advanced stages. However, in age-matched diabetic apoE−/− mice, larger, more advanced fibrous plaques with a propensity for cap formation were observed (Figure 1b). 30 Lesion complexity was defined by the presence of cholesterol clefts, necrosis, or fibrous cap formation. 30
Figure 1. Blockade of RAGE suppresses accelerated atherosclerosis in diabetic apo E−/− mice: early intervention. Apo E−/− mice were either rendered diabetic with streptozotocin or treated with citrate buffer (control). In diabetic mice, animals were treated with once daily vehicle, murine serum albumin (MSA), or different daily doses of sRAGE. After 6 weeks of diabetes or control treatment, the heart and aorta were dissected. The impact of RAGE blockade on lesion area (a) and complexity (b) is shown.
In this model, diabetes was followed by a time-dependent increase in plasma total cholesterol. 30 After 6 weeks of diabetes, ≈2-fold increase in cholesterol was noted in diabetic apoE−/− mice compared with nondiabetic animals, whereas plasma triglyceride levels were unchanged. Fractionation of plasma cholesterol by density gradient ultracentrifugation revealed significant increases in chylomicrons/very-low-density lipoprotein (≈2.6-fold) and intermediate-density lipoprotein/low-density lipoprotein (LDL) (≈2.5-fold) in age-matched diabetic versus euglycemic apoE−/− mice, whereas HDL was more modestly altered (≈1.5-fold higher in diabetic compared with control mice). 30 In contrast, density gradient ultracentrifugation showed minimal to no differences in plasma triglyceride profile. 30
In parallel with elevation of glucose, enhanced formation of AGEs in diabetic apoE−/− mice was shown by ≈2.3-fold increase in AGE-immunoreactive epitopes in acid-soluble material extracted from kidney tissue after 6 weeks of diabetes, compared with euglycemic apo E−/− controls and ≈2.5-fold increase in plasma AGEs in diabetic apoE−/− mice compared with nondiabetic control mice. 30 Further studies revealed that aortic lysates retrieved from diabetic apo E−/− mice displayed increased levels of the proinflammatory RAGE ligand family, S100/calgranulin epitopes, compared with nondiabetic apo E−/− mice of the same age. 31
It is of interest that although Kunjathoor et al did not find substantial atherosclerosis in diabetic (streptozotocin) C57BL/6 mice, 25 the trigger to atherosclerosis, dietary-induced hyperlipidemia, differed from the means to induce hyperlipidemia in apoE−/− mice. These findings highlight the concept that in addition to genetic background, other influences, such as the degree and nature of the lipid elevation, may importantly influence the development and progression of atherosclerosis in diabetes.
Accelerated Diabetic Atherosclerosis: The Role of RAGE
Consequent to the development of the streptozotocin–apo E−/− model of complex accelerated atherosclerosis in murine diabetes, the impact of blockade of RAGE was tested. In the first studies, murine-soluble RAGE (sRAGE), the extracellular two-thirds of the receptor that functions to bind ligand and thereby block interaction with and activation of cell surface RAGE, was used. 32 Soluble RAGE was administered once daily by intraperitoneal route to diabetic apo E−/− mice, commencing immediately at the time hyperglycemia developed. Compared with diabetic mice receiving mouse serum albumin (MSA), diabetic apo E−/− mice treated with sRAGE showed dose-dependent suppression of accelerated atherosclerosis (Figure 1a and 1b, respectively). In addition to diminished atherosclerotic lesion area, lesion complexity was decreased in sRAGE-treated diabetic mice. 30 This facet of the impact of RAGE blockade suggested that antagonism of this axis bore the potential to suppress lesion progression and, possibly, the development of highly inflamed unstable plaques.
Importantly, treatment of diabetic mice with sRAGE did not affect the degree of hyperglycemia or the level of insulinemia compared with diabetic mice treated with murine serum albumin. Total cholesterol and triglyceride were similarly unaffected by treatment with sRAGE, and lipid particle composition was also not changed in diabetic sRAGE-treated apoE−/− mice compared with mice receiving murine serum albumin. 30 These data suggested that the beneficial effects of blockade of RAGE were both glycemia-independent and lipid-independent, indicating that factors unique to the diabetic environment were favorably modulated. In this context, we speculate that hyperglycemia and hyperlipidemia fuel the generation of AGEs thus, sRAGE exerts its impact downstream of the traditional risk factors.
Consistent with this premise, levels of kidney and plasma AGE in diabetic mice were suppressed to levels seen in nondiabetic animals by administration of sRAGE in a dose-dependent manner. Similar results were seen on examination of plasma AGEs. 30 These findings support the contention that triggers to AGE formation might also be suppressed by RAGE blockade. To begin to address this, LDL was isolated from sRAGE-treated and murine serum albumin-treated mice. Susceptibility to copper-induced oxidation of LDL 33 retrieved from sRAGE-treated diabetic apoE−/− mice was diminished compared with diabetic mice treated with murine serum albumin, as assessed by the mean lag time to formation of conjugated dienes. 30
Blockade of RAGE: Late Intervention
To determine the potential impact of RAGE blockade in established atherosclerotic lesions, the streptozotocin-induced diabetes apo E−/− model was used. 34 Mice were rendered diabetic with streptozotocin at age 6 weeks and left untreated until age 14 weeks. At age 14 weeks, certain diabetic apo E−/− mice were euthanized to establish the “baseline” atherosclerotic lesion area/complexity. Induction of diabetes was associated with increased atherosclerotic lesion area compared with nondiabetic controls at age 14 and 20 weeks (Figure 2b and 2a and 2d and 2c, respectively). In diabetic mice treated with sRAGE from ages 14 to 20 weeks, atherosclerotic lesion area was significantly reduced compared with mice treated with murine serum albumin (Figure 2e and 2d, respectively). Oil red O-stained cross-sections of the aorta through the aortic sinus revealed that compared with nondiabetic mice, diabetic mice displayed larger and more extensive atherosclerotic lesions at 14 weeks (Figure 2f and 2g, respectively) and 20 weeks (Figure 2h and 2i, respectively). In diabetic mice treated with sRAGE, atherosclerotic lesions were smaller than those observed in vehicle-treated diabetic mice at age 20 weeks (Figure 2j and 2i, respectively).
Figure 2. Blockade of RAGE suppresses accelerated atherosclerosis in diabetic apo E−/− mice: late intervention. Apo E−/− mice were rendered hyperglycemic using streptozotocin or were treated with citrate buffer at age 6 weeks. At age 14 weeks, mice were treated with sRAGE, 100 μg per day, or murine serum albumin (MSA). Mice were euthanized at age 20 weeks and the aortae dissected and photographed (a to e). In other studies, sections at the aortic root were prepared and stained with oil red O (f to j). Scale bar: a to e, 0.3 cm f to j, 125 μm.
Quantitative morphometric analysis was performed and confirmed that diabetic mice treated with sRAGE at 20 weeks displayed a significant decrease in mean atherosclerotic lesion area compared with murine serum albumin-treated diabetic animals (Figure 3a). Although mean lesion area was increased in diabetic mice at 20 weeks versus 14 weeks, mean lesion area in sRAGE-treated diabetic mice at 20 weeks was not significantly different than that observed in diabetic mice at 14 weeks (Figure 3a). 34 In addition, at age 20 weeks, mean lesion area in sRAGE-treated diabetic mice was significantly reduced compared with lesions in nondiabetic animals of the same age (Figure 3a). 34
Figure 3. Blockade of RAGE suppresses established atherosclerosis in diabetic apo E−/− mice: lesion area and complexity. Quantification of mean atherosclerotic lesion area (a) and mean number of complex lesions per section (b) were determined.
Furthermore, we examined lesion complexity (as defined, fibrous caps, cholesterol caps, or lesion necrosis) 30 in these animals. In diabetic mice treated with sRAGE from age 14 weeks to 20 weeks, the mean number of complex lesions was significantly reduced compared with diabetic animals treated with murine serum albumin, and lesion complexity was not significantly different in sRAGE-treated diabetic mice at 20 weeks compared with diabetic mice at age 14 weeks (Figure 3b). 34 A trend toward decreased lesion complexity was observed in sRAGE-treated diabetic mice at age 20 weeks compared with nondiabetic animals of the same age, although the results did not achieve statistical significance (Figure 3b). 34 In parallel with stabilization of atherosclerotic lesion area and complexity, RAGE blockade was associated with decreased vascular expression of vascular cell adhesion molecule-1, JE-monocyte chemoattractant peptide-1, cyclooxygenase-2, nitrotyrosine epitopes, matrix metalloproteinase-9 (antigen and activity), and tissue factor epitopes (Figure 3b). 34
In addition to biochemical and molecular pathways linked to atherosclerosis, we examined collagen content within the atherosclerotic plaques using Picrosirius red staining and polarized light microscopy. Diabetes was associated with a significant increase in extent of collagen per lesion in diabetic mice versus nondiabetic controls at 20 weeks of age. In the presence of RAGE blockade, a significant decrease in collagen content per lesion was noted compared with that seen in diabetic mice treated with murine serum albumin. 34 The extent of collagen deposition in diabetic sRAGE-treated mice was not significantly different than that observed in nondiabetic animals of the same age or diabetic mice at age 14 weeks. 34 We propose that in diabetes, increased numbers of smooth muscle cells and collagen content in diabetic atherosclerotic plaques, the significant increase in numbers of mononuclear phagocytes, and the striking enhancement of proinflammatory mechanisms within the lesions tips the balance between inflammation and lesion stability. Furthermore, especially in diabetic lesions, increased matrix metalloproteinase expression/activity and generation of tissue factor antigen within the plaques enhances the likelihood of plaque progression and instability.
Certainly, these concepts require further testing in distinct species such as pigs or nonhuman primates to address the potential role of RAGE blockade in suppressing plaque instability.
Diabetes and Atherosclerosis: Other Models to Test the Impact of RAGE
It is important to note that the effects of RAGE blockade on atherosclerosis were not limited to apo E−/− mice. An additional murine model was used to trigger basal hypercholesterolemia on which to superimpose hyperglycemic stress. When mice deficient in the LDL receptor were rendered diabetic and fed a diet of normal chow, not enriched in fat, increased atherosclerotic lesion area ensued. On administration of sRAGE, accelerated atherosclerosis was attenuated. 35 These findings were in contradistinction to the work of Reavan et al. These investigators found that induction of diabetes by streptozotocin in LDL receptor−/− mice fed a high-fat diet over an extended time course did not result in acceleration of atherosclerosis. 36 We posit that the differences between these 2 study outcomes may be ascribed, at least in part, to the premise that at the aortic root, the degree of atherosclerosis that may be achieved is finite. Thus, especially over long time courses in Western diet-fed mice, differences between diabetic and nondiabetic groups may become less apparent as atherosclerosis in the nondiabetic animals continues to progress.
Furthermore, we recently extended these concepts to murine models of insulin-resistant, type 2 diabetes. To accomplish this, we bred apo E−/− mice into the db/db background. These animals, deficient in leptin receptor signaling, display striking hyperinsulinemia, hyperglycemia, and obesity. Compared with nondiabetic apo E−/− littermates, apo E−/−/db/db mice displayed increased atherosclerosis, which was decreased on daily administration of sRAGE. 37
Diabetes, Atherosclerosis, and Hyperlipidemic Mice: Effect of Distinct Interventions
The murine models of streptozotocin-induced diabetic apo E and LDL receptor−/− mice have also been used by other investigators to test the impact of distinct pathways and interventions. For example, Lin et al showed that dietary restriction of glycotoxins provides anti-atherogenic protection in streptozotocin-treated apo E−/− mice these findings provide further support for the AGE hypothesis in vascular injury. In the AGE-restricted group of diabetic apo E−/− mice, immunohistochemistry revealed decreased accumulation of AGEs and AGE receptors including RAGE, in parallel with reduced numbers of inflammatory cells and expression of tissue factor, vascular cell adhesion molecule-1, and JE-monocyte chemoattractant peptide-1 in the vascular lesions. 38 Levi et al have shown that treatment of diabetic LDL receptor−/− mice with peroxisome proliferator-activated gamma agonists such as rosiglitazone attenuates atherosclerosis in these animals, without impacting on levels of blood glucose. 39 The role of angiotensin-converting enzyme was studied by Candido et al these investigators showed that administration of perindopril for 20 weeks prevented diabetes-associated atherosclerosis in parallel with reduced aortic expression of angiotensin-converting enzyme, connective tissue growth factor, and vascular cell adhesion molecule-1 overexpression. 40 In other studies, Tse et al tested the impact of 17 β-estradiol in diabetic male apo E−/− mice and demonstrated that chronic treatment with 17 β-estradiol reduced lesion area in multiple vascular segments of these mice, in parallel with decreased blood glucose and levels of triglyceride. 41
Thus, taken together, such mouse models of diabetic atherosclerosis provide a template for dissection of molecular pathways linked to the pathogenesis of macrovascular complications of diabetes and, also, the potential impact of therapeutic interventions.
Diabetes and the Problem of Restenosis
Epidemiologic studies indicate that in human subjects with diabetes, exaggerated restenosis commonly accompanies acute arterial injury, such as that induced by therapeutic angioplasty. 42–44 Studies further demonstrated that stenting of the treated vessel does not offer full protection from restenosis and coronary events. 42,43 Even with the recent development of sirolimus-coated stents, it is not yet clear if diabetic subjects will fully benefit from this novel therapy. 45–47
To dissect the biochemical and molecular events linked to enhanced restenosis in diabetic human subjects, it was first necessary to establish animal models to readily address these concepts.
Studies in Rats
The first studies using diabetic rats were performed in obese Zucker rats displaying type 2 insulin-resistant diabetes. 48 Interestingly, compared with induction of diabetes (type 1) in Sprague-Dawley rats by streptozotocin in which neointimal hyperplasia was not exaggerated, Park et al showed that obese Zucker rats subjected to carotid balloon injury displayed a >2-fold increase in neointimal area compared with lean Zucker (nondiabetic) rats on day 21 after injury. 48 In the intima of these animals, cell proliferation markedly increased, commencing at day 3 and persisting through day 14. This animal model was then used to test the role of RAGE blockade. Administration of sRAGE was begun just before arterial injury and was continued through the first week after balloon injury. Compared with vehicle treatment (albumin), sRAGE-treated rats displayed a significant decrease in neointimal expansion. 49 A key finding in this study was the marked reduction in incorporation of bromodeoxyuridine in the smooth muscle cells of the expanding neointima in sRAGE-treated rats. These experiments strongly suggested that RAGE-expressing smooth muscle cells, at least in part, contributed importantly to diabetes-associated enhanced neointimal expansion after acute injury.
Studies in Murine Models
These findings suggested that it was logical to further dissect the role of RAGE in arterial injury. In rat models, however, the inability to use transgenic/knockout approaches limited this approach. Furthermore, direct studies in murine models would facilitate the testing of genetically RAGE-modified mice, thereby supporting the premise that the chief target of sRAGE was the receptor itself. Thus, to use murine models, acute femoral artery endothelial injury was induced according to a previously-established and validated model. 50 Guide wire-induced injury to the femoral artery was performed and the impact of both pharmacological and genetic modification of RAGE was tested. RAGE and its ligands, CML-AGEs and S100/calgranulins, were upregulated in the femoral vessel wall after injury, particularly in smooth muscle cells. 51 On day 28 after injury, a dose-dependent decrease in intima/media (I/M) ratio was observed in mice treated with sRAGE versus vehicle (murine serum albumin). 51 Importantly, homozygous RAGE−/− mice displayed decreased I/M ratio on day 28 after injury compared with littermate control animals subjected to arterial denudation (Figure 4a through 4c). Numbers of proliferating smooth muscle cells were significantly reduced in RAGE−/− mice versus controls. 51
Figure 4. Homozygous RAGE (0) mice and transgenic mice expressing signaling deficient dominant negative (DN) RAGE in smooth muscle cells display decreased neointimal expansion consequent to acute arterial injury. Homozygous RAGE−/− mice (a to c) and transgenic mice expressing DN RAGE selectively in smooth muscle cells (driven by the SM22 alpha promoter) (d) and wild-type littermates were subjected to femoral artery guide wire injury. Intima/media ratio was determined on day 28 after injury (a, d). Von Gieson elastic stain was performed on a representative femoral artery section from a wild-type RAGE-bearing (b) and a RAGE−/− mouse (c). Scale bar: 50 μm.
As homozygous RAGE−/− mice do not express RAGE in any cell type, an additional strategy was used to specifically dissect the role of smooth muscle cell RAGE in modulating neointimal expansion on acute arterial injury. Previous studies showed that deletion of the cytosolic domain of RAGE imparted a dominant-negative (DN) effect when wild-type RAGE-bearing vascular or mononuclear phagocyte-like cells were transfected with constructs encoding DN RAGE, a DN effect ensued on incubation with RAGE ligands such as S100B or CML-AGE. 3,9 Because smooth muscle cells were the principal RAGE-expressing cells in the expanding neointima, 51 these concepts were tested in transgenic mice expressing DN RAGE specifically in smooth muscle cells driven by the SM22α promoter. Compared with wild-type littermates, transgenic SM22 DN RAGE mice subjected to arterial injury displayed a significant decrease in I/M ratio on day 28 (Figure 4d). Thus, these experiments provided further support for roles for RAGE in neointimal expansion. In addition to pharmacological blockade of RAGE, genetically modified RAGE mice (deletion or interruption of smooth muscle cell signal transduction) displayed decreased injury-triggered neointimal expansion.
However, a limitation of inducing arterial injury in C57BL/6 mice is that there is little evidence of inflammatory cell influx into the injured vessel wall in wild-type animals. To address this concept, femoral artery injury was induced in apo E−/− mice based on the premise that basal vascular perturbation secondary to hyperlipidemia would augment the response to arterial injury, including migration of inflammatory cells. We found that I/M ratios were higher, reflecting enhanced neointimal expansion in apo E−/− mice vessels 28 days after injury versus C57BL/6 mice (≈2-fold higher I/M ratio). 51 In euglycemic apo E−/− mice, administration of sRAGE diminished I/M ratio on day 28. In parallel, numbers of mononuclear phagocytes infiltrating the injured artery were also reduced. 51 Thus, apo E−/− mice may provide a superior system to test the role of smooth muscle cell and mononuclear phagocyte RAGE in the response to acute arterial injury.
In addition, these studies highlighted the role of RAGE-dependent Jak/Stat signaling in smooth muscle cells. In femoral vessels after injury, although increased phosphorylation of ERK 1/2, protein kinase B (akt), and Jak2/Stat3 were observed versus sham treatment, sRAGE appeared to only exert its effects on Jak2/Stat3 signaling. 51
Diabetes and Restenosis: Other Models and Distinct Interventions
Diabetic rodent models of arterial injury have also been tested for the impact of distinct interventions on neointimal expansion. Phillips et al used Western diet-fed apo E−/− mice to show that these mice develop insulin-resistant diabetes administration of rosiglitazone prevented the development of hyperglycemia and hyperinsulinemia. 52 In parallel, neointimal expansion was reduced by 65% after arterial injury in Peroxisome proliferator-activated gamma agonist (rosiglitazone) versus vehicle-fed apo E−/− mice. Macrophage infiltration into the expanding neointima was also reduced by this intervention. 52 Note that these studies are in contradistinction to those of Levi et al 39 with respect to the effect of rosiglitazone on hyperglycemia. Specifically, in Levi’s studies, apo E−/− mice were made frankly diabetic by relative deficiency of insulin using streptozotocin 39 in the Phillips studies, hyperglycemia was induced by feeding Western diet, thereby causing insulin resistance. 54
In other studies, Min et al administered troglitazone to Otsuka Long-Evans Tokushima fatty rats and found that smooth muscle cell proliferation was markedly reduced, in parallel with decreased neointimal expansion after carotid balloon injury. 53 The role of dietary glycotoxins (AGEs) on neointimal expansion was directly tested in apo E−/− mice by Lin et al. Mice were randomized to high-AGE or low-AGE diet (the latter with 10-fold lower AGE content) and subjected to femoral artery injury. In parallel with decreased neointimal expansion in the low-AGE diet-fed mice, AGE content was reduced in the injured vessel wall. 54
The complex nature of murine/rodent models in dissection of the biologic response to arterial injury superimposed on chronic hyperglycemia was underscored by the studies of Stephenson et al. 55 These investigators reported that db/db mice displayed markedly decreased neointimal expansion consequent to femoral artery endoluminal wire injury compared with wild-type controls. 55 Four hours after acute arterial injury, medial smooth muscle death was reduced in db/db versus wild-type mice. However, smooth muscle cell proliferation did not appear to differ between db/db versus wild-type mice. 55 These findings suggest important roles for leptin in modulation of stimulated smooth muscle cell properties. Oda et al reported that leptin stimulated phosphorylation and activation of MAP kinase and phosphatidylinositol 3-kinase activity in cultured smooth muscle cell one consequence of which was increased smooth muscle cell proliferation and migration. 56 Taken together, these considerations underscore the complexity of these model systems and suggest that preclinical testing of novel therapeutic targets in restenosis ultimately requires the use of species such as pigs, rabbits, or nonhuman primates before testing in human subjects. 57,58
The ultimate goal of our studies is to determine the potential role of RAGE blockade as a therapeutic strategy in the macrovascular complications of diabetes. Thus, the development of key models to test the role of the receptor in a time-specific and cell-specific manner has provided a template for in-depth dissection of the role of RAGE in vascular pathology. The finding that blockade of RAGE affords benefit in mice bearing both insulin-deficient and insulin-resistant diabetes further supports the role of this receptor in hyperglycemic states. These considerations underscore the concept that the products of the downstream effects of high glucose and oxidant stress generate species that are signal transduction ligands of the receptor.
It is important to stress that RAGE-dependent mechanisms in vascular stimulation are not limited to diabetes. As the vasculature in euglycemic atherosclerotic plaques is also susceptible to oxidant stress, inflammation, and, thus, generation of AGEs, albeit to lesser degrees, it was logical to test the impact of RAGE blockade. Thus, it was not surprising that administration of sRAGE to euglycemic apo E−/− mice from ages 14 to 20 weeks attenuated atherosclerotic lesion area and complexity, without an impact on lipid levels. 34
In conclusion, these findings underscore the usefulness of rodent, and particularly mouse, models in dissection of the mechanisms linking RAGE to accelerated atherosclerosis and restenosis, particularly in diabetes. Propelled by compelling data in animal models, we propose that testing of RAGE blockade in a multi-targeted approach in diabetes may lead to a successful reduction of the macrovascular complications of types 1 and 2 diabetes. Clinical trials to address these issues in human subjects are on the horizon.
REGULATION OF RAGE BY MODULATION OF PREASSEMBLY
As outlined above, preassembly of RAGE is required for activation and downstream signaling. The formation of multimers is favored by high surface concentrations of RAGE on the cell surface. The data available helped to develop a model for the initiation of the signal cascade by RAGE/ligand interaction. However, there is only rare data about the mechanism stopping the signal. There is evidence that several mechanisms control the surface concentration of signal-competent RAGE, and a few potential mechanisms are outlined below.
Receptor internalization after activation is a well-known mechanism to shut down signaling of an active receptor/ligand complex. Internalization of S100 proteins, binding to RAGE into granular structures, has been observed already by Hsieh et al. . Immunofluorescence and histochemical staining of different cell types revealed RAGE in the cytoplasmic membrane and intracellularly, in vesicular or granular structures [56, 157– 162]. Later on, it was shown that the RAGE/S100B complex is internalized via lipid rafts [163, 164]. Likewise, the RAGE/AGE complex was internalized and was monitored in vesicular structures  (Fig. 3, lower).
On the other hand, the surface concentration of a receptor can be increased immediately by fusion of vesicles containing the receptor with the cytoplasmic membrane. Receptor endocytosis and recycling are described for a plethora of receptors and are a tightly regulated process. Dysregulation of receptor trafficking can lead to severe disorders, including cancer [166–168]. Evidence for vesicle-mediated exposure of RAGE on the endothelial surface comes from studies by Frommhold et al. [63, 64]. The authors observed that upon a proinflammatory stimulus, RAGE is presented within a few minutes on the endothelium, acting as a counter-receptor for leukocytes that trigger adhesion and transmigration. The almost-immediate appearance of RAGE on the surface indicates that no de novo synthesis of RAGE has occurred but rather, that RAGE was mobilized from internal stores. Such a transport of RAGE to the surface is in agreement with the observation of intracellular vesicular structures harboring RAGE. Such vesicles might be directed to the cytoplasmic membrane and fuse there, resulting in a fast increase in surface concentration of RAGE.
A further option to modulate RAGE signaling is the production and secretion of soluble RAGE species (esRAGE, sRAGE) by alternative splicing or receptor shedding. Like the full-length receptor, soluble RAGE can bind RAGE ligands and block RAGE signaling effectively. In most studies, it is assumed that soluble RAGE acts as a decoy receptor, competing with full-length RAGE for ligands. The levels of soluble RAGE species have to be tightly controlled, as changes in the level of soluble RAGE are associated with diabetes, cardiovascular disorders, and certain types of cancer [148, 169, 170].
However, the levels of soluble RAGE remain rather low compared with full-length RAGE , raising the question of how the low amounts of extracellular soluble RAGE could compete effectively with full-length RAGE. We propose that soluble RAGE does not act as a simple competitor but attenuates activation of full-length RAGE by disturbing the preassembly of the receptor on the cell surface. It has been shown that RAGE–RAGE interactions occur via the V and C1 domain, enabling sRAGE to interact with membrane-bound, full-length RAGE. The resulting heteromultimers would be not signaling-competent, as sRAGE is devoid of the cytoplasmic domain. This model is corroborated by studies using a deletion mutant of RAGE that contains the transmembrane helix but lacks the cytoplasmic domain. This RAGE variant has been shown to exert a dominant-negative effect on RAGE signaling. Coexpression of this cytoplasmic deletion mutant with full-length RAGE could abrogate RAGE signaling, although intact receptor molecules are present. This dominant-negative phenotype is explained readily by the formation of nonfunctional heterodimers between the truncated and full-length form of RAGE. It is reasonable to suppose that soluble RAGE, devoid of membrane and cytoplasmic domain, forms similar signaling-incompetent heteromultimers with full-length RAGE (Fig. 3, lower).
Two further alternative splicing variants expressed at observable levels show deletions or insertions in the extracellular ligand-binding domain and are, therefore, not able to bind RAGE ligands . As homophilic RAGE interactions are mediated as well as via the C1 domain, it is very likely that these variants are still able to form heterodimers with full-length RAGE. Correspondingly, these receptor multimers may not be activated, as the ligands bind only to a fraction of the membrane-bound molecules and fail to stabilize a signal-competent complex.
Three major findings were obtained in the present study. First, RAGE, S100A8 and S100A9 were overexpressed in IPAH and HPAH-PASMCs in the absence of any external growth stimulus. Second, PDGF-BB up-regulated the expression of RAGE in IPAH and HPAH-PASMCs. Third, AS-1, an inhibitor of RAGE signaling, significantly inhibited overgrowth under a condition of no growth stimulation and PDGF-stimulated proliferation of IPAH and HPAH-PASMCs. RAGE plays an important role in the inappropriate increase of PAH-PASMCs, and its inhibition may be a new therapeutic strategy for PAH.
Several investigators have reported that expression levels of RAGE were increased in serum or plasma, small pulmonary arteries and PASMCs of patients with PAH [9, 19, 20]. Here, we showed that RAGE was overexpressed in PASMCs of not only patients with IPAH but also patients with HPAH including patients with BMPR2 mutation and SMAD9 mutation. Therefore, RAGE is a common exacerbating factor in PAH. As for ligands for RAGE, Greenway et al. reported that S100A4/Mts1 was expressed in smooth muscle cells of lesions showing neointimal formation and with increased intensity in vessels with an occlusive neointima and plexiform lesions of patients with pulmonary hypertension secondary to congenital heart disease . Meloche et al. reported that activation of RAGE by S100A4 decreased BMPR2 expression in human PASMCs . We showed that S100A8 and A9 were also overexpressed in IPAH and HPAH-PASMCs. We previously reported that S100A8/A9 induced phosphorylation of RAGE and led to activation of diverse signal effectors such as Rac1, Akt, p38, JNK, IKK, and NFkB in HEK293 and 293-hMD2-CD14 cells . Further studies are needed to clarify the precise role of S100A8 and A9 in the pathogenesis of PAH.
We previously reported that PDGF-BB stimulation caused a higher growth rate of cultured PASMCs from patients with IPAH than that of control cells [3, 10]. Here we showed that PDGF-BB also up-regulated the expression of RAGE in IPAH and HPAH-PASMCs.
We have recently reported that Toll/IL-1 receptor (TIR) domain-containing adaptor protein (TIRAP) transduces RAGE signaling and that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions . A synthetic low-molecular-weight TIR/BB-Loop mimetic, AS-1, inhibits TIR domain-mediated signaling . AS-1 finally inhibits RAGE signaling. AS-1, an inhibitor of RAGE signaling, significantly inhibited overgrowth under a condition of no growth stimulation and PDGF-stimulated proliferation of IPAH and HPAH-PASMCs.
This study was performed in a small population of patients. Therefore, we could not perform quantitative analysis of staining. This is a study limitation. Further study is needed.
In conclusion, RAGE plays a crucial role in the inappropriate increase of PAH-PASMCs. Inhibition of RAGE signaling may be a new therapeutic strategy for PAH.