Evaluation of translocator protein quantification as a tool for characterising macrophage burden in human carotid atherosclerosis.

Macrophage presence within atherosclerotic plaque is a feature of instability and a risk factor for plaque rupture and clinical events. Activated macrophages express high levels of the translocator protein/peripheral benzodiazepine receptor (TSPO/PBR). In this study, we investigated the potential for quantifying plaque inflammation by targeting this receptor. TSPO expression and distribution in the plaque were quantified using radioligand binding assays and autoradiography. We show that cultured human macrophages expressed 20 times more TSPO than cultured human vascular smooth muscle cells (VSMCs), the other abundant cell type in plaque. The TSPO ligands [3H](R)-1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H](R)-PK11195) and [3H]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([3H]-DAA1106) bound to the same sites in human carotid atherosclerotic plaques in vitro, and demonstrated significant correlation with macrophage-rich regions. In conclusion, our data indicate that radioisotope-labelled DAA1106 has the potential to quantify the macrophage content of atherosclerotic plaque.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification.

Vascular smooth muscle cells (VSMCs) exhibit an extraordinary capacity to undergo phenotypic change during development, in vitro and in association with disease. Unlike other muscle cells they do not terminally differentiate. Development and maintenance of the mature contractile phenotype is regulated by a number of interacting transcription factors. In response to injury contractile VSMCs can be induced to change phenotype, proliferate and migrate to effect repair. On completion of the repair process VSMCs return to a nonproliferating contractile phenotype. In this way, in the context of atherosclerosis, a protective fibrous cap is formed and maintained at sites of injury. However in disease, when modulatory signals are perturbed, this phenotypic transition is dysregulated and VSMCs are induced to undergo inappropriate differentiation into cells with features of other mesenchymal lineages such as osteoblasts, chondrocytes and adipocytes. Moreover, evidence is accumulating that these aberrant phenotypic transitions contribute to the pathogenesis of vascular diseases such as atherosclerosis and Monckeberg's Sclerosis. Indeed, the osteo/chondrocytic conversion of VSMCs and the association of this phenotype with vascular calcification is a paradigm for how inappropriate differentiation can influence disease processes. Understanding of the mechanisms and signalling pathways involved in this particular phenotype change is well advanced offering the possibility for the design of successful therapeutic interventions in the future.

Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis.

BACKGROUND: Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. METHODS AND RESULTS: In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase-polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased approximately 3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. CONCLUSIONS: These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.

Imaging atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron emission tomography.

BACKGROUND: Atherosclerotic plaque rupture is usually a consequence of inflammatory cell activity within the plaque. Current imaging techniques provide anatomic data but no indication of plaque inflammation. The glucose analogue [18F]-fluorodeoxyglucose (18FDG) can be used to image inflammatory cell activity non-invasively by PET. In this study we tested whether 18FDG-PET imaging can identify inflammation within carotid artery atherosclerotic plaques. METHODS AND RESULTS: Eight patients with symptomatic carotid atherosclerosis were imaged using 18FDG-PET and co-registered CT. Symptomatic carotid plaques were visible in 18FDG-PET images acquired 3 hours post-18FDG injection. The estimated net 18FDG accumulation rate (plaque/integral plasma) in symptomatic lesions was 27% higher than in contralateral asymptomatic lesions. There was no measurable 18FDG uptake into normal carotid arteries. Autoradiography of excised plaques confirmed accumulation of deoxyglucose in macrophage-rich areas of the plaque. CONCLUSIONS: This study demonstrates that atherosclerotic plaque inflammation can be imaged with 18FDG-PET, and that symptomatic, unstable plaques accumulate more 18FDG than asymptomatic lesions.

Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions.

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.

A polymorphism of the human matrix gamma-carboxyglutamic acid protein promoter alters binding of an activating protein-1 complex and is associated with altered transcription and serum levels.

Matrix gamma-carboxyglutamic acid protein (MGP) is a mineral-binding extracellular matrix protein synthesized by vascular smooth muscle cells (VSMCs) and chondrocytes that is thought to be a key regulator of tissue calcification. In this study, we identified four polymorphisms in the promoter region of the human MGP gene. Transfection studies showed that the G-7A and T-138C polymorphisms have an important impact on in vitro promoter activity when transiently transfected into VSMCs. We found that one of these polymorphisms (T-138C) is significantly correlated with serum MGP levels in human subjects. Promoter deletion analysis showed that this polymorphism lies in a region of the promoter critical for transcription in VSMCs. This region contains a potential activating protein-1 (AP-1) binding element located between -142 and -136. We have demonstrated that the T-138C polymorphism results in altered binding of an AP-1 complex to this region. The -138T allelic variant binds AP-1 complexes consisting primarily of c-Jun, JunB and its partners Fra-1 and Fra-2 in rat VSMC. Furthermore, the -138T variant form of the promoter was induced following phorbol 12-myristate 13-acetate treatment, while the -138C variant was refractive to phorbol 12-myristate 13-acetate treatment, confirming that AP-1 factors preferentially bind to the -138T variant. This study therefore suggests that a common polymorphism of the MGP promoter influences binding of the AP-1 complex, which may lead to altered transcription and serum levels. This could have important implications for diseases such as atherosclerosis and aortic valve stenosis, since it strongly suggests a genetic basis for regulation of tissue calcification.

Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions.

Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17), and the CX(3)C chemokine fractalkine (CX(3)CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions.

Defect in insulin-like growth factor-1 survival mechanism in atherosclerotic plaque-derived vascular smooth muscle cells is mediated by reduced surface binding and signaling.

Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.

Transcriptional regulation of matrix gla protein.

Matrix Gla Protein (MGP) is a small protein which is thought to be an inhibitor of tissue calcification and a regulator of cell differentiation. In this study we have examined the transcriptional regulation of MGP within rat vascular smooth muscle cells (VSMCs). We found that MGP transcription is downregulated by retinoic acid and transforming growth factor beta (TGF beta) whereas it is upregulated by vitamin D3 and cyclic AMP.

The role of apoptosis in the initiation of vascular calcification.

The initiation sites for calcification in cartilage and bone are cellular products called matrix vesicles. Similar structures have been found in calcified arteries and recent studies suggest that these may be derived from apoptotic cells. It is well established that there is a link between cell death and calcification but the mechanism involved is not known. Since apoptotic cell death is known to occur in the vasculature, we set out to investigate the role of apoptosis in the initiation of vascular calcification. We used a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Our studies revealed that apoptosis occurred prior to the onset of calcification and that VSMC "blebs" or apoptotic bodies (ABs) could concentrate calcium in a crystallised form. These observations suggest that apoptosis is involved in the development of VSMC calcification and that VSMC-derived ABs have similarities with matrix vesicles.

Inflammatory mechanisms.

Traditional concepts of the pathogenesis of acute coronary syndromes have changed over the last few years. In particular it has been demonstrated that high-risk lesions are not necessarily angiographically severe. Rather, unstable high risk lesions are the ones composed of large lipid cores and thin fibrous caps. It is now widely accepted that plaque instability is related to the development of inflammation within the intima. A consequence of this is that stabilization of lesions provides a new therapeutic target. Furthermore, there is growing evidence that statins may stabilize lesions by altering the inflammatory response. A brief overview of these developments and their impact on clinical practice is presented.

Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues.

In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as 'dystrophins of the nucleus' to maintain nuclear organization and structural integrity.

Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Matrix gla protein is regulated by a mechanism functionally related to the calcium-sensing receptor.

Matrix Gla protein (MGP) is a mineral binding extra-cellular matrix protein which is thought to be a key inhibitor of tissue and vascular calcification. It is known to be upregulated in areas of extracellular calcification possibly to limit further harmful calcification. In this study we have demonstrated that extracellular ionic calcium (high levels of which induce calcification) is a key signal for MGP regulation and that this effect is mediated by a G protein mediated cation-sensing mechanism, functionally related to, but molecularly distinct from the calcium-sensing receptor. We therefore propose that this novel cation sensing mechanism may play a homeostatic role in preventing pathological calcification.