In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

Non-enzymatic glycation of extracellular matrix with (U-13C5)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3-CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.

Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation.

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing.

Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs.

Neuropathy and the vascular-bone axis in diabetes: lessons from Charcot osteoarthropathy.

Emerging evidence from the last two decades has shown that vascular calcification (VC) is a regulated, cell-mediated process orchestrated by vascular smooth muscle cells (VSMCs) and that this process bears many similarities to bone mineralization. While many of the mechanisms driving VSMC calcification have been well established, it remains unclear what factors in specific disease states act to promote vascular calcification and in parallel, bone loss. Diabetes is a condition most commonly associated with VC and bone abnormalities. In this review, we describe how factors associated with the diabetic milieu impact on VSMCs, focusing on the role of oxidative stress, inflammation, impairment of the advanced glycation end product (AGE)/receptor for AGE system and, importantly, diabetic neuropathy. We also explore the link between bone and VC in diabetes with a specific emphasis on the receptor activator of nuclear factor κß ligand/osteoprotegerin system. Finally, we describe what insights can be gleaned from studying Charcot osteoarthropathy, a rare complication of diabetic neuropathy, in which the occurrence of VC is frequent and where bone lysis is extreme.

The implementation of an integrated information system for substance use screening in general medical settings.

UNLABELLED: The Massachusetts Screening, Brief Intervention and Referral to Treatment (MASBIRT) Program, a substance use screening program in general medical settings, created a web-based, point-of-care (POC), application--the MASBIRT Portal (the "Portal") to meet program goals. OBJECTIVES: We report on development and implementation of the Portal. METHODS: Five year program process outcomes recorded by an independent evaluator and an anonymous survey of Health Educator's (HEs) adoption, perceptions and Portal use with a modified version of the Technology Readiness Index are described. [8] Specific management team members, selected based on their roles in program leadership, development and implementation of the Portal and supervision of HEs, participated in semi-structured, qualitative interviews. RESULTS: At the conclusion of the program 73% (24/33) of the HEs completed a survey on their experience using the Portal. HEs reported that the Portal made recording screening information easy (96%); improved planning their workday (83%); facilitated POC data collection (84%); decreased time dedicated to data entry (100%); and improved job satisfaction (59%). The top two barriers to use were "no or limited wireless connectivity" (46%) and "the tablet was too heavy/bulky to carry" (29%). Qualitative management team interviews identified strategies for successful HIT implementation: importance of engaging HEs in outlining specifications and workflow needs, collaborative testing prior to implementation and clear agreement on data collection purpose, quality requirements and staff roles. DISCUSSION: Overall, HEs perceived the Portal favorably with regard to time saving ability and improved workflow. Lessons learned included identifying core requirements early during system development and need for managers to institute and enforce consistent behavioral work norms. CONCLUSION: Barriers and HEs' views of technology impacted the utilization of the MASBIRT Portal. Further research is needed to determine best approaches for HIT system implementation in general medical settings.

The effects of anterior load carriage on lower limb gait parameters during obstacle clearance.

The purpose of this study was to assess the effect of anterior load carriage on obstacle-crossing behaviour, with a focus on lower limb gait parameters. Nine male participants (age 23+/-1.8 years, height 176+/-5.0cm) volunteered. Participants either walked without a load (No Load), or carried a load (2KG (empty box), 5KG, 10KG), and stepped over a 20cm obstacle. Vision of the obstacle was obscured 1.0m to 1.3m prior to the obstacle. Significant correlations were found between trail limb toe distance and lead limb toe clearance, in the 2KG, 5KG, and 10KG conditions. Toe clearance increased with load (No Load, 147.3+/-13.9mm; 2KG, 162.5+/-15.6mm; 5KG, 167.6+/-17.6mm; 10KG, 173.9+/-17.5mm; p<0.0001). Trail limb toe distance, trail limb toe distance variability, lead heel distance variability, and lead limb toe clearance variability were greater in the 2KG, 5KG, and 10KG conditions, compared with the No Load condition. Participants adopted a conservative gait pattern during obstacle crossing when carrying a load, evidenced by increasing toe clearance, which may have been influenced by availability of visual information regarding obstacle position. In contrast with previous literature, increased lead limb toe clearance may have been associated with absence of relative surface height difference pre- and post-obstacle crossing.

Role for alkaline phosphatase as an inducer of vascular calcification in renal failure?

Vascular calcification is associated with increased cardiovascular morbidity and mortality. A number of calcification inhibitors have been defined recently, including inorganic pyrophosphate (PP(i)), an important physicochemical inhibitor of hydroxyapatite crystal growth. Increased hydrolysis of PP(i) by tissue-nonspecific alkaline phosphatase (TNAP) may occur in renal failure and act to enhance mineralization of vessels.

Exploring the biology of vascular calcification in chronic kidney disease: what's circulating?

Chronic kidney disease (CKD) is associated with fatal cardiovascular consequences in part due to ectopic calcification of soft tissues particularly arteries, capillaries, and cardiac valves. An increasing body of evidence from experimental studies and in vivo data suggest that (I) a mineral imbalance with hyperphosphatemia and high-circulating calcium x phosphate product, (II) a deficiency of systemic or local calcification inhibitors, (III) death or 'damage' of vascular smooth muscle cells (VSMCs), and/or (IV) phenotypic transformation of VSMCs to osteo/chondrocytic cells may all act in concert to initiate and sustain vascular calcification. In CKD patients inhibitory systems are overwhelmed by a multitude of agents that induce VSMC damage and cell death resulting in the release of vesicles capable of nucleating basic calcium phosphate. Studies with genetically altered mice have identified both local and systemic calcification inhibitors that act to maintain VSMC differentiation or regulate vesicle properties. However, for many of these proteins the mechanisms and sites of action are still under investigation. In particular, it is unclear whether factors present in the circulation have an inhibitory role there and whether circulating levels of these proteins influence or are indicative of underlying disease processes in individual patients. A greater understanding of the origins and roles of potential circulating inhibitors may result in novel strategies aimed at the prevention or reversal of the life-limiting calcifying vasculopathies seen in CKD patients.

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 calcification and osteoporosis--from clinical observation towards molecular understanding.

Patients with osteoporosis frequently suffer from vascular calcification, which was shown to predict both cardiovascular morbidity/mortality and osteoporotic fractures. Various common risk factors and mechanisms have been suggested to cause both bone loss and vascular calcification, including aging, estrogen deficiency, vitamin D and K abnormalities, chronic inflammation and oxidative stress. Major breakthroughs in molecular and cellular biology of bone metabolism and the characterization of knockout animals with deletion of bone-related genes have led to the concept that common signaling pathways, transcription factors and extracellular matrix interactions may account for both skeletal and vascular abnormalities. For example, mice that lack the cytokine decoy receptor osteoprotegerin or the hormone Klotho display a combined osteoporosis-arterial calcification phenotype. In this review, we summarize the current data and evaluate potential mechanisms of the osteoporosis-arterial calcification syndrome. We propose a unifying hypothesis of vascular calcification that combines both active and passive mechanisms of vascular mineralization with aspects of bone resorption and age-related changes.

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.

Mechanisms of vascular calcification in renal disease.

Vascular calcification is commonplace in patients with end-stage renal disease where it develops rapidly and predicts a variety of adverse outcomes. The processes responsible for vascular calcification have been the focus of much research, aided in recent decades by molecular genetic techniques and in vitro models. Converging evidence now suggests that vascular calcification is an active, regulated process, with abundant similarities to the process of skeletal mineralization. Using an in vitro model of calcifying vascular smooth muscle cells (VSMCs), we have shown that a mineral imbalance induces VSMC apoptosis, and that VSMC apoptotic bodies and vesicles can nucleate basic calcium phosphate in the form of hydroxyapatite, the same mineral found in bone. Gene expression studies suggest that the normal vessel wall expresses proteins such as matrix Gla protein that inhibit calcification. In addition, circulating proteins such as fetuin-A are produced at remote sites and act to inhibit soft tissue calcification systemically. However, down-regulation or perturbation of these proteins may lead to a phenotypic transformation of VSMCs to osteo/chondrocytic-like cells while the calcified environment may stimulate macrophages to adopt osteoclastic properties. Both clinical and basic research findings indicate an inverse relationship between bone mineralization and vascular calcification. The mechanisms linking these two processes are a topic for further investigation, with current theories proposing a role for lipids, common regulatory molecules, and calcium and bone turnover. We have synthesized these findings into a theoretical model offering a putative pathway for the development of severe vascular calcification in end-stage renal disease.

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.

Biology of calcification in vascular cells: intima versus media.

BACKGROUND: Vascular calcification occurs at two distinct sites within the vessel wall: the intima and the media. Intimal calcification occurs in the context of atherosclerosis, associated with lipid, macrophages and vascular smooth muscle cells, whereas medial calcification can exist independently of atherosclerosis and is associated with elastin and vascular smooth muscle cells. PATHOGENESIS: In this review we compare intimal and medial calcification, particularly discussing the mechanisms which may be responsible for each type of calcification. Similar mechanisms probably initiate and regulate both forms of calcification including the generation of matrix vesicles/apoptotic bodies and local expression of mineralization-regulating proteins. However, since different modifying agents such as lipids in the intima and elastin in the media are present at the sites of calcification and are associated with particular diseases, this implies that the etiologies of these processes differ. For example, intimal calcification is associated with atherosclerosis while medial calcification occurs commonly in the diabetic neuropathic leg. CLINICAL IMPORTANCE: Since both types of calcification correlate with significant morbidity and mortality, we discuss the different types of calcification in terms of their clinical importance.

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.

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.

A potential role for sterol 27-hydroxylase in atherogenesis.

OBJECTIVE: 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells. RESULTS: Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core. CONCLUSIONS: 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.