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Trion A, van der Laarse AVascular smooth muscle cells and calcification in atherosclerosis. Am Heart 147:808-814
Abstract
Vascular calcification is a prominent feature of atherosclerosis but the mechanisms underlying vascular calcification are still obscure. Since bone-associated proteins such as osteonectin, osteocalcin, and matrix Gla protein have been detected in calcified vascular tissues, calcification has been considered to be an organized, regulated process similar to mineralization in bone tissue. Vascular smooth muscle cells (VSMCs) are currently considered to be responsible for the formation of vascular calcifications. Apoptosis of VSMCs appears to be a key factor in this process, while other factors including cell-cell interactions (macrophages and VSMCs), lipids, and plasma inorganic phosphate levels modulate the calcification process. The focus of this review is on the role of VSMCs in the development of calcifications in atherosclerotic plaques.
... SMCs produce extracellular matrix components of the plaque fibrous cap, which stabilizes the plaque and prevents rupture (13). Later in plaque progression, some SMCs transform into osteogenic cells and contribute to plaque calcification (14), which correlates with risk of plaque rupture in humans (15). Finally, some SMCs within the plaque have recently been found to dedifferentiate and become inflammatory-like, losing expression of smooth muscle α-actin and instead expressing traditionally leukocyte-specific markers, even acting as foam cells themselves [reviewed in Bennett et al. (16)]. ...
... Fasting glucose (mg/dL) 94.1 ± 7.2 (14) 105.3 ± 11.4 (15) 145.5 ± 18.6 (15) 102.6 ± 8.9 ...
... 8.16 ± 0.19 (14) Spleen weight (mg) 110.0 ± 3.3 ...
Objective: Elevated levels of the hormone aldosterone are associated with increased risk of myocardial infarction and stroke in humans and increased progression and inflammation of atherosclerotic plaques in animal models. Aldosterone acts through the mineralocorticoid receptor (MR) which is expressed in vascular smooth muscle cells (SMCs) where it promotes SMC calcification and chemokine secretion in vitro. The objective of this study is to explore the role of the MR specifically in SMCs in the progression of atherosclerosis and the associated vascular inflammation in vivo in the apolipoprotein E knockout (ApoE−/−) mouse model.
Methods and Results: Male ApoE−/− mice were bred with mice in which MR could be deleted specifically from SMCs by tamoxifen injection. The resulting atheroprone SMC-MR-KO mice were compared to their MR-Intact littermates after high fat diet (HFD) feeding for 8 or 16 weeks or normal diet for 12 months. Body weight, tail cuff blood pressure, heart and spleen weight, and serum levels of glucose, cholesterol, and aldosterone were measured for all mice at the end of the treatment period. Serial histologic sections of the aortic root were stained with Oil Red O to assess plaque size, lipid content, and necrotic core area; with PicroSirius Red for quantification of collagen content; by immunofluorescent staining with anti-Mac2/Galectin-3 and anti-smooth muscle α-actin antibodies to assess inflammation and SMC marker expression; and with Von Kossa stain to detect plaque calcification. In the 16-week HFD study, these analyses were also performed in sections from the brachiocephalic artery. Flow cytometry of cell suspensions derived from the aortic arch was also performed to quantify vascular inflammation after 8 and 16 weeks of HFD. Deletion of the MR specifically from SMCs did not significantly change plaque size, lipid content, necrotic core, collagen content, inflammatory staining, actin staining, or calcification, nor were there differences in the extent of vascular inflammation between MR-Intact and SMC-MR-KO mice in the three experiments.
Conclusion: SMC-MR does not directly contribute to the formation, progression, or inflammation of atherosclerotic plaques in the ApoE−/− mouse model of atherosclerosis. This indicates that the MR in non-SMCs mediates the pro-atherogenic effects of MR activation.
... Both type 2 bone morphogenetic protein (BMP-2) and osteopontin have been found in human atherosclerotic plaque, where they have been recognized as hallmarks of calcification and inflammatory destabilization of the plaque [22][23][24][25]. Oxidative stress promotes vascular cell calcification in vitro, effectively inhibited by the use of antioxidant factors such as ω-3 fatty acid and high-density lipoproteins [26]. ...
... Murine models of atherosclerotic calcification have been developed to further understand the underlying mechanism involved in their development. Spontaneous vascular calcification has been founding several genetically different inbred mouse strains, including C5BL/6, Balb/C, C3H/HeJ, DBA/2J, SM/J, and MRL-lpr/lpr mice, and found to increase following a high-fat/high-cholesterol diet [26]. ...
Cardiovascular calcification is a multifactorial and complex process involving an array of molecular mechanisms eventually leading to calcium deposition within the arterial walls. This process increases arterial stiffness, decreases elasticity, influences shear stress events and is related to an increased risk of morbidity and mortality associated with cardiovascular disease. In numerous in vivo and in vitro models, warfarin therapy has been shown to cause vascular calcification in the arterial wall. However, the exact mechanisms of calcification formation with warfarin remain largely unknown, although several molecular pathways have been identified. Circulating miRNA have been evaluated as biomarkers for a wide range of cardiovascular diseases, but their exact role in cardiovascular calcification is limited. This review aims to describe the current state-of-the-art research on the impact of warfarin treatment on the development of vascular calcification and to highlight potential molecular targets, including microRNA, within the implicated pathways.
... These different locations represent discrete pathophysiological processes (9,31 32). Intimal calcification is an active process similar to bone formation and occurs by expressing growth factors, matrix proteins, and other bonerelated proteins (33). The process is related to inflammatory cells, lipid deposits, and vascular smooth muscle cells. ...
... Dale et al. stated that in the mammographic examination, women with myocardial infarction (MI) history had significantly higher frequencies of BAC than women without a history of MI. (12). Besides, another study determined an association between mammographically detected arterial calcifications and ASCVD (33). Otherwise, to the conclusions of their research, Ak et al. expressed that BAC did not help determine coronary atherosclerosis (45). ...
Background:
Breast arterial calcification (BAC) is easily detected and commonly observed on screening mammography. That is more frequent among people with diabetes, and these people are at risk of coronary artery disease. The incidence of BAC increases with advancing age. We aimed to determine whether BAC detected by mammography is associated with the development of coronary atherosclerosis in asymptomatic women. It can help reduce morbidity and mortality secondary to atherosclerotic cardiovascular disease.
Methods:
We included one hundred and eighty women over the age of 40 who underwent mammography screening in this multi-modality study. Mammography evaluated the presence of calcifications, the number of involved arteries, and the distribution. We questioned the patients about cardiovascular risk factors such as hypertension and diabetes. The coronary artery disease severity was assessed according to both Agatston and calcium scores on coronary computed tomography (CT). Besides, the relationship between these scores and correlation with carotid artery intima-media thickness was investigated. We stated mean and standard deviation (SD) for continuous variables and reported frequency distributions and percentages. SPSS software version 25.0 was used to perform the analysis.
Results:
Overall, 302 of 3,600 cases were positive for BAC. However, 120 of them could be included in the study by the eligibility criteria of our research. In univariate analysis, age, hyperlipidemia, DM, HT, and smoking history were risk factors that significantly affected BAC development. The impact of age and diabetes were maintained in the logistic regression analysis (P<0.005), while the significant effect of the other variables was vanished (P>0.02). Furthermore, moderate and high BAC scores were correlated with higher coronary atherosclerosis scores.
Conclusions:
BAC may predict an additional risk factor for coronary artery disease, particularly in patients having higher scores. That may be an accurate indicator for subsequent development of coronary arterial calcifications so that it may be possible to reduce morbidity and mortality associated with coronary atherosclerosis.
... Vascular smooth muscle cells (SMCs) are essential for regulating blood pressure and maintaining and remodeling the extracellular matrix (ECM) of blood vessels. [8] SMCs dysfunction is involved in the occurrence and development of cardiovascular disease and considerable studies have suggested that SMCs play a key role in vascular calcification [9]. SMCs-mediated calcification involves with the phenotypic transition of SMCs into osteoblast-like cells, development of calcifiable ECM as well as cell apoptosis and vesicle release. ...
... For instance, excessive proliferation of SMCs contributes to atherosclerotic lesion formation and postangioplasty restenosis. [9] In addition, SMCs can transform into a spectrum of phenotypes in response to local stimulation, driving vascular disease development [8]. In healthy adult tissue, SMCs have a mature and contractile phenotype, but they retain remarkable phenotypic plasticity as they are not terminally differentiated [29,30]. ...
Vascular stiffening occurs with advanced age and under pathological conditions such as vascular calcification, during which the osteogenesis of smooth muscle cells (SMCs) plays a key role. However, whether the stiffness of cellular microenvironment influences osteogenic responses in vascular SMCs is not well understood. Here, we cultured SMCs on the poly(dimethylsiloxane) (PDMS) substrates with varying stiffness from 0.363 to 2.327 MPa. The cell osteogenic transdifferentiation was induced by β-glycerophosphate. Our findings demonstrated that the extent of osteogenesis in SMCs varied with the substrate stiffness. On three substrate stiffness, cells on the intermediate one (0.909 MPa) showed the highest extent of the osteogenesis based on the expression of osteogenic markers and calcium deposition. Transforming growth factor-β1 and autophagy were involved in this stiffness-dependent process. This work highlights the importance of substrate stiffness to the osteogenesis of vascular SMCs, giving new scientific information for understanding of SMCs-mediated vascular calcification and designing of vascular implants.
... Nutrients 2020, 12, 2777 2 of 13 and mortality prompted by cardiovascular disease [6]. Vascular calcification is a process that is similar with the bone formation process [1] and is initiated by phenotype change of vascular smooth muscle cells (VSMCs) to osteoblast-like cells, followed by deposition of hydroxyapatite [7]. Physiologically normal VSMCs maintain blood flow by regulating the contraction and relaxation of vessels [8]. ...
... The vessel that has osteoblast-like cells demonstrated an increased vascular stiffness, which leads to reducing blood flow [8]. The osteoblast-like VSMCs secreted bone-related protein biomarkers involving osteogenesis such as osteoblast differentiation and maturation [7][8][9]. Bone morphogenetic protein-2 (BMP2) is increased during vascular calcification and leads to the activation of core-binding factor alpha-1 (CBFα-1), a principal transcriptional regulator of the maturation of osteoblasts in the bone [10][11][12]. CBFα-1 also increased the expression of osteoblast proteins in VSMCs and led to phenotype changes to osteoblast-like cells [13]. ...
Advanced glycation end products/receptor for AGEs (AGEs/RAGEs) or Toll like receptor 4 (TLR4) induce vascular smooth muscle cell (VSMC) phenotype changes in osteoblast-like cells and vascular calcification. We analyzed the effect of Ecklonia cava extract (ECE) or pyrogallol-phloroglucinol-6,6-bieckol (PPB) on VSMC phenotype changes and vascular calcification prompted by a high-fat diet (HFD). HFD unregulated RAGE, TLR4, transforming growth factor beta (TGFβ), bone morphogenetic protein 2 (BMP2), protein kinase C (PKC), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signals in the aorta of mice. ECE and PPB restored the increase of those signal pathways. AGE- or palmitate-treated VSMC indicated similar changes with the animal. HFD increased osteoblast-like VSMC, which was evaluated by measuring core-binding factor alpha-1 (CBFα-1) and osteocalcin expression and alkaline phosphatase (ALP) activity in the aorta. ECE and PPB reduced vascular calcification, which was analyzed by the calcium deposition ratio, and Alizarin red S stain was increased by HFD. PPB and ECE reduced systolic, diastolic, and mean blood pressure, which increased by HFD. PPB and ECE reduced the phenotype changes of VSMC to osteoblast-like cells and vascular calcification and therefore lowered the blood pressure.
... The contractile phenotype is characterized by differentiation markers that are grouped into early, mid-term, and late differentiation markers [49]. On the other hand, synthetic VSMCs secrete many ECM proteins, including osteopontin and osteonectin [50,51]. ...
... In response to inflammation, VSMCs reduce the expression of these markers and adopt the expression of synthetic dedifferentiated phenotype [98]. VSMCs then become active in secreting ECM molecules such as osteopontin and osteonectin [51,99,100]. As such, high levels of these proteins indicate VSMC switch to synthetic phenotype. ...
Remodeling of arterioles is a pivotal event in the manifestation of many inflammation-based cardio-vasculopathologies, such as hypertension. During these remodeling events, vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. The latter is characterized by increased proliferation, migration, and invasion. Compounds with anti-inflammatory actions have been successful in attenuating this phenotypic switch. While the vast majority of studies investigating phenotypic modulation were undertaken in VSMCs isolated from large vessels, little is known about the effect of such compounds on phenotypic switch in VSMCs of microvessels (microVSMCs). We have recently characterized a novel homoisoflavonoid that we called 7-O-methylpunctatin (MP). In this study, we show that MP decreased FBS-induced cell proliferation, migration, invasion, and adhesion. MP also attenuated adhesion of THP-1 monocytes to microVSMCs, abolished FBS-induced expression of MMP-2, MMP-9, and NF-κB, as well as reduced activation of ERK1/2 and FAK. Furthermore, MP-treated VSMCs showed an increase in early (myocardin, SM-22α, SM-α) and mid-term (calponin and caldesmon) differentiation markers and a decrease in osteopontin, a protein highly expressed in synthetic VSMCs. MP also reduced transcription of cyclin D1, CDK4 but increased protein levels of p21 and p27. Taken together, these results corroborate an anti-inflammatory action of MP on human microVSMCs. Therefore, by inhibiting the synthetic phenotype of microVSMCs, MP may be a promising modulator for inflammation-induced arteriolar pathophysiology.
... Atherosclerotic intimal calcification, accompanied by cholesterol deposition, has been associated with atherosclerotic occlusive lesions. This process is the predominant type of calcification observed in the aorta, coronary and peripheral arteries [4,5] and is correlated with the presence of vascular smooth muscle cells (VSMCs), as well as macrophages in lipid-rich atherosclerotic plaques [6][7][8]. Atherosclerotic intimal calcification is associated with classic cardiovascular risk factors like age, male sex, smoking, hypertension, dyslipidemia, diabetes mellitus as well as newer ones, such as inflammation. It is focal and the adjacent vascular wall may be free of lesions. ...
Vascular calcification (VC) is a complex process of calcium deposition on the arterial wall and atherosclerotic plaques and involves interaction between vascular smooth muscle cells, inflammatory and VC mediators. The latter are independent predictors of cardiovascular morbidity and mortality and potential targets of pharmaceutical therapy. This paper is a narrative review of the complex mechanisms of VC development and in this context the potential anti-atherosclerotic effects of statins. At the initial stages of atherosclerosis VC correlates with atherosclerosis burden and in the long-term with cardiovascular morbidity and mortality. A plethora of animal and clinical studies have proposed statins as the cornerstone of primary and secondary prevention of atherosclerotic cardiovascular disease. Based on coronary computed tomography data, high doses of statins may have negligible or even positive effects on the progression of coronary artery calcification. Growing data support an increase in atherosclerotic plaque calcification in peripheral arteries (e.g., carotids), after long-term, statin-therapy. Despite the paradox of increasing VC, those effects of statins have been associated with higher plaque stability, reducing the risk of consequent adverse events. Statins seem to promote a “favorable” atherosclerotic calcification, suppressing atherosclerotic lesion expansion and their vulnerability. More studies are required to clarify the underlying mechanisms.
... The process of vascular calcification is similar to that of osteogenesis and is caused by the transformation of VSMCs into the osteoblast-like phenotype. Osteoblast-like VSMCs secrete bone-related protein biomarkers, which are involved in osteoblast differentiation, maturation, and other osteogenic processes, ultimately leading to calcification of the intima and plaque formation [40][41][42]. However, the osteoblast-like phenotype transformation of VSMCs can increase plaque stability during the late stage of AS, and prevent plaque rupture, which has positive implications. ...
Cardiovascular disease is one of the leading causes of human mortality worldwide, mainly due to atherosclerosis (AS), and the phenotypic transition of vascular smooth muscle cells (VSMCs) is a key event in the development of AS. Exosomes contain a variety of specific nucleic acids and proteins that mediate intercellular communication. The role of exosomes in AS has attracted attention. This review uses the VSMC phenotypic transition in AS as the entry point, introduces the effect of exosomes on AS from different perspectives, and discusses the status quo, deficiencies, and potential future directions in this field to provide new ideas for clinical research and treatment of AS.
... Moreover, as explained in the introduction, these cells are typically calcium-laden in PAD. Thus, to model advanced calcified lesion in PAD, RAOSMC were cultured and maintained in a black-walled, clear bottom, tissue-culture treated plates with growth medium compared to calcification medium for two weeks [33]. Growth medium contained DMEM with 10% fetal calf serum. ...
Peripheral artery disease (PAD) is a systemic vascular disease of the legs that results in a blockage of blood flow from the heart to the lower extremities. Now one of the most common causes of mortality in the U.S., the first line of therapy for PAD is to mechanically open the blockages using balloon angioplasty. Coating the balloons with antiproliferative agents can potentially reduce vessel re-narrowing, or restenosis after surgical intervention, but current drug-coated balloons releasing chemotherapy agents like paclitaxel have in some cases shown increased mortality long-term. Our aim was to design a novel drug-coated balloon using a polymeric nanodelivery system for a sustained release of polyphenols that reduce restenosis but with reduced toxicity compared to chemotherapy agents. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles with entrapped quercetin, a dimethoxy quercetin (rhamnazin), as well as quercetin covalently attached to PLGA, were developed. Balloon catheters were coated with polymeric nanoparticles using an ultrasonic method, and nanoparticle characteristics, drug loading, coating uniformity and drug release were determined. The adhesion of nanoparticles to vascular smooth muscle cells and the antiproliferative effect of nano-delivered polyphenols were also assessed. Of the nanoparticle systems tested, those with covalently attached quercetin provided the most sustained release over a 6-day period. Although these particles adhered to cells to a smaller extent compared to other nanoparticle formulations, their attachment was resistant to washing. These particles also exhibited the greatest anti-proliferative effect. In addition, their attachment was not altered when the cells were grown in calcifying conditions, and in PAD tissue calcification is typically a condition that impedes drug delivery. Moreover, the ultrasonic coating method generated a uniform balloon coating. The polymeric nanoparticle system with covalently attached quercetin developed herein is thus proposed as a promising platform to reduce restenosis post-angioplasty.
... The release of proinflammatory cytokines and chemokines, along with vascular smooth muscle cell (VSMC) migration and extracellular matrix secretion, contribute to the formation of the fibrous cap that encapsulates the atheroma [92]. The presence of vascular calcification is a strong indicator of advanced atherosclerosis, whereby hydroxyapatite crystals form in the vessel walls, reducing arterial elasticity and altering atherosclerotic plaque stability [93,94]. Microcalcifications present in the fibrous cap of atherosclerotic lesions cause local stress that leads to cap rupture [95]. ...
Pleckstrin homology-like domain, family A, member 1 (PHLDA1) is a multifaceted intracellular protein belonging to the evolutionarily conserved pleckstrin homology-related domain family. Its murine homologue, T-cell death-associated gene 51 (TDAG51), was initially discovered for its role in activation-induced apoptosis in T-cell hybridomas. In recent years, PHLDA1 has received increased attention due to its association with obesity, fatty liver disease, diabetes, atherosclerosis, and cancer. Accumulating evidence also supports its role in endoplasmic reticulum stress signaling pathways as a crucial mediator of apoptosis, autophagy, and cell proliferation. In this review, the current knowledge of PHLDA1 gene and protein regulation, localization, and function is summarized. This review highlights the pro- and anti-apoptotic roles of PHLDA1 that contribute to metabolic disease.
... Atherosclerosis (AS) is a severe vascular disorder causing myocardial infarction, stroke, and gangrene [1] and usually accompanied by the accumulation of endothelial damages, proinflammation factors, and vascular smooth muscle cells (VSMCs) [2,3]. VSMC proliferation and neointima formations are the dominant events in the development of atherosclerotic lesions [4,5]. Increasing evidence indicates that noncoding RNAs, including microRNAs (miRNAs) with length of ~20 nucleotides and long noncoding RNAs (lncRNAs) with lengths over 200 nucleotides, play pivotal roles in the process of atherosclerosis [6][7][8][9][10][11]. Circular RNAs (circRNAs) are a family of special lncRNAs with covalently closed continuous loops formed by the 3' and 5' ends. ...
Atherosclerosis is a severe vascular disorder causing myocardial infarction, stroke, and gangrene. Circular RNA Testis-expressed 14 (hsa_circ_0107197, CircTEX14) is a newly discovered circRNA that may have a critical role in the pathogenesis of atherosclerosis. Here, we aimed to further explore the exact role of circRNA TEX14 in the cardiovascular system. Serum samples of atherosclerosis patients (n = 48) and healthy volunteers (n = 48) were collected to assess circTEX14 expressions. Quantitative reverse transcription-PCR (qRT-PCR), cell proliferation assay, migration assay, cell necrosis assay, Annexin staining, TUNEL assays, RNA immunoprecipitation (RIP) assays, dual-luciferase reporter assays, wound healing assays, and Western blot were performed to examine the roles of circTEX14, miR-6509-3p, and thanatos-associated domain-containing apoptosis-associated protein 1 (THAP1) in ox-LDL-stimulated vascular smooth muscle cells (VSMCs). We found that circTEX14 expressions were decreased and miR-6509-3p expressions were increased in the serum samples of atherosclerosis patients and ox-LDL-stimulated VSMCs. CircTEX14 overexpression inhibited proliferation and migration and enhanced apoptosis of VSMCs. CircTEX14 suppressed miR-6509-3p expressions through direct interaction. MiR-6509-3p or THAP1 knockdown reversed the effects of circTEX14 overexpression on proliferation, migration, and apoptosis of ox-LDL-stimulated VSMCs. In conclusion, circTEX14 inhibited proliferation and enhanced apoptosis via modulating miR-6509-3p/THAP1 in ox-LDL-stimulated VSMCs and might be a useful target for atherosclerosis treatment.
... It was reported that ROS-mediated oxidative damage was associated with HPi-induced calcification (Trion and van der Laarse, 2004;Choi et al., 2017). Therefore, HPi-induced accumulation of ROS and superoxide anion, and DNA damage were all detected in MOVAS cells. ...
Vascular calcification was an independent risk of cardiovascular and cerebrovascular diseases (CCDs). Studies reported that conditioned media of choroid plexus epithelium cells (CPECs-CM) showed potential neuroprotective effects. However, the protective effect of CPECs-CM against vascular calcification (VC) has not been reported yet. Herein, high phosphate (HPi)–induced calcification model in mouse aortic vascular smooth muscle cells (MOVAS) was established, and the protective effects and underlying mechanism of CPECs-CM against HPi-induced calcification were explored. The results indicated that CPEC cells were successfully isolated and cultured, and CPECs-CM co-treatment significantly inhibited HPi-induced calcification of MOVAS cells through blocking alkaline phosphatase activity and expression. CPECs-CM co-treatment also suppressed reactive oxide species–mediated DNA damage in HPi-treated MOVAS cells. Moreover, dysfunction of MAPKs and PI3K/AKT pathways both contributed to HPi-induced calcification of MOVAS cells, and CPECs-CM co-treatment attenuated HPi-induced calcification by normalizing MAPKs and PI3K/AKT expression. Taken together, our findings provide evidence that CPECs-CM had the potential to inhibit vascular calcification with potent application in chemoprevention and chemotherapy of human CCD.
... Advanced stages of atherosclerosis are characterized by vascular calcification [125]. Notably, ROS, namely H 2 O 2 , have the potential to promote calcification by inducing the VSMC switch to an osteoblast-like cell phenotype in a defined osteogenic medium [126]. ...
Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism in the cell. At physiological levels, they play a vital role in cell signaling. However, high ROS levels cause oxidative stress, which is implicated in cardiovascular diseases (CVD) such as atherosclerosis, hypertension, and restenosis after angioplasty. Despite the great amount of research conducted to identify the role of ROS in CVD, the image is still far from being complete. A common event in CVD pathophysiology is the switch of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. Interestingly, oxidative stress is a major contributor to this phenotypic switch. In this review, we focus on the effect of ROS on the hallmarks of VSMC phenotypic switch, particularly proliferation and migration. In addition, we speculate on the underlying molecular mechanisms of these cellular events. Along these lines, the impact of ROS on the expression of contractile markers of VSMCs is discussed in depth. We conclude by commenting on the efficiency of antioxidants as CVD therapies.
... Of note, in calcific aortic stenosis, the smooth muscle cell phenotype remains, and in myofibroblast differentiation and calcification, Runx2 and α-SMA are dually increased in the osteogenic/osteoblastic phenotype (37,38). As multiple sub-populations of VSMCs exist, including synthetic, contractile, and particularly calcifying prone cell phenotypes, we recommend other sources of VSMCs are investigated with ucOCN to confirm our findings (39,40). Lastly, it may transpire that cOCN, which has a higher affinity for calcium ions, may be more relevant in vascular calcification and further studies should also address this specifically. ...
Osteocalcin (OCN) is a bone-derived protein that is detected within human calcified vascular tissue. Calcification is particularly prevalent in chronic kidney disease (CKD) patients but the role of OCN in calcification, whether active or passive, has not been elucidated. Part 1: The relationship between OCN, CKD and vascular calcification was assessed in CKD patients (n = 28) and age-matched controls (n = 19). Part 2: in vitro, we analyzed whether addition of uncarboxylated osteocalcin (ucOCN) influenced the rate or extent of vascular smooth muscle cell (VSMC) calcification. Human aortic VSMCs were cultured in control media or mineralisation inducing media (MM) containing increased phosphate with or without ucOCN (10 or 30 ng/mL) for up to 21 days. Markers of osteogenic differentiation and calcification were determined [alkaline phosphatase (ALP) activity, total intracellular OCN, Runx2 expression, α-SMA expression, alizarin red calcium staining, and calcium quantification]. Part 1 results: In our human population, calcification was present (mean age 76 years), but no differences were detected between CKD patients and controls. Plasma total OCN was increased in CKD patients compared to controls (14 vs. 9 ng/mL; p < 0.05) and correlated to estimated glomerular filtration rate (p < 0.05), however no relationship was detected between total OCN and calcification. Part 2 results: in vitro, ALP activity, α-SMA expression and calcium concentrations were significantly increased in MM treated VSMCs at day 21, but no effect of ucOCN was observed. Cells treated with control media+ucOCN for 21 days did not show increases in ALP activity nor calcification. In summary, although plasma total OCN was increased in CKD patients, this study did not find a relationship between OCN and calcification in CKD and non-CKD patients, and found no in vitro evidence of an active role of ucOCN in vascular calcification as assessed over 21 days. ucOCN appears not to be a mediator of vascular calcification, but further investigation is warranted.
... ON is known for the affinity for hydroxyapatite and collagen and is involved in bone development [277]. ON is present in calcified vascular tissues suggesting that VC is a process similar to bone tissue mineralization [278]. Increased ON levels have been shown to inhibit cell spreading and cell attachment in cultured endothelial cells, disrupt focal adhesions in fibroblasts and increase the production and activity of matrix metalloproteinases leading to matrix degradation in some cell types [279]. ...
Over the last decades, the association between vascular calcification (VC) and all-cause/cardiovascular mortality, especially in patients with high atherogenic status, such as those with diabetes and/or chronic kidney disease, has been repeatedly highlighted. For over a century, VC has been noted as a passive, degenerative, aging process without any treatment options. However, during the past decades, studies confirmed that mineralization of the arteries is an active, complex process, similar to bone genesis and formation. The main purpose of this review is to provide an update of the existing biomarkers of VC in serum and develop the various pathogenetic mechanisms underlying the calcification process, including the pivotal roles of matrix Gla protein, osteoprotegerin, bone morphogenetic proteins, fetuin-a, fibroblast growth-factor-23, osteocalcin, osteopontin, osteonectin, sclerostin, pyrophosphate, Smads, fibrillin-1 and carbonic anhydrase II.
... These isolated cells started to behave like osteoblast cells by secreting bone-related protein biomarkers including transcription factors for osteoblast differentiation and maturation (4)(5)(6). Increased reactive oxygen species, myeloperoxidase activity and pro-inflammatory cytokines such as IL-1, IL-6, TNF- and minimally oxidized LDL are some of the factors/substances so far identified for inducing vascular calcification (7,8). ...
The role of oxidized high- density lipoprotein (oxHDL) and the protective effects of adiponectin in terms of vascular calcification is not well-established. This study was conducted to investigate the effects of oxHDL with regard to inflammation and vascular calcification and to determine the protective role of adiponectin in attenuating the detrimental effects of oxHDL. Cell viability, mineralization, and calcification assays were conducted to optimize the concentration of oxHDL. Then, human vascular smooth muscle cells (HAoVSMCs) were incubated with β-glycerophosphate, HDL, oxHDL, adiponectin, or the combination of oxHDL with adiponectin for 24 h. Protein expression of IL-6, TNF-α, osterix, RUNX2, ALP, type 1 collagen, osteopontin, osteocalcin, WNT-5a, NF-ĸβ(p65), cAMP and STAT-3 were measured by ELISA kits. OxHDL induced vascular calcification by promoting the formation of mineralization nodules and calcium deposits in HAoVSMCs. This was accompanied by an increased secretion of IL-6, osterix, WNT-5a and NF-ĸβ (p65). Interestingly, these detrimental effects of oxHDL were suppressed by adiponectin. Besides, incubation of adiponectin alone on HAoVSMCs showed a reduction of inflammatory cytokines, osteoblastic markers (RUNX2, osterix and osteopontin), WNT-5a and NF-ĸβ (p65). This study exhibits the ability of oxHDL in inducing inflammation and vascular calcification and these detrimental effects of oxHDL can be attenuated by adiponectin.
... Deposits of calcium phosphate crystals form in distinct layers on the arterial wall and are associated with specific pathologies. For example, calcification is frequently observed in atherosclerotic lesions (intimal calcification) [7], which is associated with vascular smooth muscle cells (VSMCs) and macrophages in lipidrich areas of atheromatous plaques [8]. By contrast, medial calcification, also known as "Monckeberg's medial sclerosis", occurs in the elastic region of the arteries, is almost exclusively associated with VSMCs, and is common in arteriosclerosis related to aging, diabetes, and CKD [9]. ...
Cardiovascular complications due to accelerated atherosclerosis and arterial stiffening are the leading cause of morbidity and mortality in the Western society. Both pathologies are frequently associated with vascular calcification. Deposits of calcium phosphate salts, mainly in form of hydroxyapatite, is the hallmark of vascular calcification. Calcification is frequently observed in atherosclerotic lesions (intimal calcification), associated with vascular smooth muscle cells (VSMCs) and macrophages. By contrast, medial calcification, occurring in the elastic region of the arteries, is almost exclusively associated with VSMCs, and is common in arteriosclerosis related to aging, diabetes, and chronic kidney disease. In extracellular fluids, a range of endogenous low- and high-molecular weight calcification inhibitors are present, including osteopontin, matrix-Gla proteins and Fetuin A. Moreover, pyrophosphate deficiency plays a key role in vascular calcification. Pyrophosphate is produced by extracellular hydrolysis of ATP and is degraded to phosphate by tissue non-specific alkaline phosphatase. Loss of function in the enzymes and transporters involved in the extracellular pyrophosphate metabolism leads to excessive deposition of calcium-phosphate salts. This review summarizes the current knowledge about endogenous mechanisms of protection against calcification in the aortic wall, focusing on the role of extracellular pyrophosphate metabolism in vascular smooth muscle cells and macrophages.
... Neointimal hyperplasia is a pathological process that often occurs after surgical intervention in vascular diseases such as pulmonary arterial hypertension and is the main contributor to post-intervention restenosis. 1 It is characterized by the excess proliferation and migration of vascular smooth muscle cells (VSMCs), promoting the development of cardiovascular diseases with cytopathological manifestations such as vascular stenosis and calcification. [2][3][4] VSMCs exist in and can interconvert between two phenotypic states, namely the contractile (differentiation) and synthetic (dedifferentiation) states. 4,5 The transformation from a contractile state to a synthetic one leads to enhanced VSMC proliferation and migration, secretion and synthesis of extracellular matrices, and formation of neointimal membranes, which is the key step in the initiation of severe vascular proliferative diseases. ...
Objectives:
Vascular disorders are associated with phenotypical switching of vascular smooth muscle cells (VSMCs). We investigated the effect of bone morphogenetic protein (BMP)-2 in controlling VSMC phenotype and vascular disorder progression. Lysine (K)-specific demethylase 1A (KDM1A) has been identified to target BMP-2 and is employed as a therapeutic means of regulating BMP-2 expression in VSMCs.
Materials and methods:
VSMCs were stimulated with angiotensin II, and the expression of KDM1A and BMP-2 was detected. VSMC proliferation, apoptosis, and phenotype were evaluated. An in vivo aortic injury model was established, and VSMC behaviour was evaluated by the expression of key markers. The activation of BMP-2-associated signalling pathways was examined.
Results:
We confirmed the inhibitory effect of KDM1A on BMP-2 activity and demonstrated that KDM1A inhibition prevented VSMC transformation from a contractile to synthetic phenotype. In angiotensin II-treated VSMCs, KDM1A inhibition triggered a decrease in cell proliferation and inflammatory response. In vivo, KDM1A inhibition alleviated post-surgery neointimal formation and collagen deposition, preventing VSMCs from switching into a synthetic phenotype and suppressing disease onset. These processes were mediated by BMP-2 through canonical small mothers against decapentaplegic signalling, which was associated with the activation of BMP receptors 1A and 1B.
Conclusions:
The regulatory correlation between KDM1A and BMP-2 offers insights into vascular remodelling and VSMC phenotypic modulation. The reported findings contribute to the development of innovative strategies against vascular disorders.
... In contrary to the traditional view, VC is now regarded as an automatic, regulatable and revisable procedure controlled by multiple factors [3]. Particularly, previous researchers have proven that apoptosis of vascular smooth muscle cells (VSMCs) exist extensively in calcified aorta [4]. In addition, several pathological studies have found that apoptotic bodies released from VSMCs can initiate VC [5]. ...
Fibroblast growth factor 21 (FGF21), a hormone with multiple metabolic properties, has proven to be pleiotropic biological effects and may play pivotal role in numerous cardiovascular and metabolic diseases in the future. Vascular calcification (VC) is a concomitant pathological process of various cardiovascular and metabolic diseases. However, the effects of FGF21 on VC remain unclear. Therefore, in this research, we aimed to explore the roles and mechanisms of FGF21 in VC induced by vitamin D3 plus nicotine (VDN) treatment rats. After 28 days VDN treatment, the calcium overload was confirmed by blood pressure, ultrasound imaging, calcium content, ALP activity and aortic pathological characteristics. In terms of FGF21, exogenous FGF21 can ameliorate the elevation of blood pressure, aortic calcification and related injury in VC rats. To investigate the mechanisms of FGF21 on VC, the endoplasmic reticulum stress (ERS) mediated apoptosis pathways were tested. As a method to detect apoptosis, the increased positive TUNEL staining cells were alleviated by FGF21 treatment. Furthermore, exogenous FGF21 can suppress the increased ERS chaperone, GRP78, in the calcified aortas. In the three pathways of ERS mediated apoptosis, we found CHOP pathway and caspase-12 pathway were involved in the treatment of FGF21, but not p-JNK/JNK pathway. Our study proved for the first time that FGF21 can inhibit the progress of VC by alleviating ERS mediated apoptosis in rats. FGF21 might be a new target for preventing and treating VC.
... For example, these VSMCs synthesize higher amounts of col- lagen type I, III and V, elastin and glycosaminoglycan, particularly chondroitin sulfate A/C and dermatan sulfate [6,110]. In addition, phenotypically-modulated VSMCs synthesize ECM mol- ecules, which are typical for osteoblasts and are involved in matrix calcification, for example, osteopontin and osteonectin [111,112]. These VSMCs also showed increased expression and Figure 6. ...
... These proteins are involved in the balance of coagulation due to the expression of various VKDPs in different tissues, and they serve a certain function, such as balancing coagulation and exhibiting an antagonistic effect to maintain homeostasis in the human body. For example, when ectopic calcification occurs, VSMCs differentiate into osteoblast-like cells (61), which produce OC to increase OC expression in the atherosclerotic vessel wall (127)(128)(129). MGP has been reported to protect blood vessels and prevent ectopic calcification, while carboxylated OC promotes calcium deposition in the bone under normal circumstances (17,130,131). ...
In postmenopausal women and elderly men, bone density decreases with age and vascular calcification is aggravated. This condition is closely associated with vitamin K2 deficiency. A total of 17 different vitamin K‑dependent proteins have been identified to date. Vitamin K‑dependent proteins are located within the bone, heart and blood vessels. For instance, carboxylated osteocalcin is beneficial for bone and aids the deposition of calcium into the bone matrix. Carboxylated matrix Gla protein effectively protects blood vessels and may prevent calcification within the vascular wall. Furthermore, carboxylated Gla‑rich protein has been reported to act as an inhibitor in the calcification of the cardiovascular system, while growth arrest‑specific protein‑6 protects endothelial cells and vascular smooth muscle cells, resists apoptosis and inhibits the calcification of blood vessels by inhibiting the apoptosis of vascular smooth muscle cells. In addition, periostin may promote the differentiation, aggregation, adhesion and proliferation of osteoblasts. Periostin also occurs in the heart and may be associated with the reconstruction of heart function. These vitamin K‑dependent proteins may exert their functions following γ‑carboxylation with vitamin K, and different vitamin K‑dependent proteins may exhibit synergistic effects or antagonistic effects on each other. In the cardiovascular system with vitamin K antagonist supplement or vitamin K deficiency, calcification occurs in the endothelium of blood vessels and vascular smooth muscle cells are transformed into osteoblast‑like cells, a phenomenon that resembles bone growth. Both the bone and cardiovascular system are closely associated during embryonic development. Thus, the present study hypothesized that embryonic developmental position and tissue calcification may have a certain association for the bone and the cardiovascular system. This review describes and briefly discusses several important vitamin K‑dependent proteins that serve an important role in bone and the cardiovascular system. The results of the review suggest that the vascular calcification and osteogenic differentiation of vascular smooth muscle cells may be associated with the location of the bone and cardiovascular system during embryonic development.
... Byon et al. described oxidative stress as an inducer of key osteochondrogenic factors that in turn promote a phenotypic switch of contractile SMCs to an osteochondrogenic phenotype [5]. Therefore, vascular SMCs contribute to the development of atherosclerotic lesions through increased migration, secretion of matrix components, osteochondrogenic differentiation, and the associated calcification [25]. Most of our results can be considered part of this causal nexus. ...
Background: In the current considerations of the genesis of cardiovascular degeneration, reactive oxygen species (ROS) have come into focus as a potential key factor. Several models of nutritive and enzymatic approaches have tried to analyze its causative role; however, they are accompanied by systemic effects and unforeseeable interaction patterns. Objective: This study analyzed the photodynamic reaction (PDR) regarding its potential to initiate a focal, non-systemic impact of oxidative stress. The induced processes and results were characterized by comparing them with the current perception of atherosclerotic vascular degeneration. Material and methods: A PDR was applied to the infrarenal aorta of rats by the combination of rose bengal and a low-power laser. The rats in the ROS and control groups (each n?= 5) where killed at six points of time. All specimens were analyzed regarding gross morphological changes as well as detailed cell composition and the dynamics of cell migration. Results: Contrary to the controls, the ROS groups displayed an initial thrombus formation that disappeared not later than at t?= 8?days. After a period of morphological latency accompanied by a significantly increased matrix metalloproteinase (MMP) activity, the ROS-exposed area displayed a significant thickening of the media (p?< 0,001) as well as a significant focal calcification (p?< 0.01). This was accompanied by a focally reduced alpha-smooth muscle actin (alpha-SMA) expression. Conclusion: The PDR induces morphological short-term effects that disappear after not later than 8 days. After a latency period, certain aspects of an atherosclerotic degeneration were obvious. In contrast, key aspects, such as increased inflammation and foam cells were missing. Nevertheless, the established concept is a suitable model to investigate the role of ROS in vascular degeneration. © 2018 Springer Medizin Verlag GmbH, ein Teil von Springer Nature
... The degradation media was diluted with fresh media in order to obtain the desired concentrations of 10, 25, 50, and 100 mg/mL. These concentrations were chosen based on work by Ref. 30 which showed significant primary vascular SMC toxicity with pure iron degradation products at 50 mg/mL. Therefore, the purpose of this experiment was to compare pure iron and galfenol cytotoxicity. ...
The gold standard of care for coronary artery disease, a leading cause of death for in the world, is balloon angioplasty in conjunction with stent deployment. However, implantation injuries and long‐term presence of foreign material often promotes significant luminal tissue growth, leading to a narrowing of the artery and severely restricted blood flow. A promising method to mitigate this process is the use of biodegradable metallic stents, but thus far they have either degraded too slowly (iron) or disappeared prematurely (magnesium). The present work investigates the use of a unique type of magnetic material, galfenol (iron‐gallium), for postoperative wireless control of stent degradation rates. Due to its magnetoelastic property, galfenol experiences longitudinal micron‐level elongations when exposed to applied magnetic fields, allowing generation of a microstirring effect that affect its degradation behavior. In vitro indirect cytotoxicity tests on primary rat aortic smooth muscle cells indicated that galfenol byproducts must be concentrated approximately seven times from collected 60 day degradation medium to cause ∼15% of death from all cells. Surface and cross‐sectional characterization of the material indicate that galfenol (Fe80Ga20) degradation rates (∼0.55% per month) are insufficient for stenting applications. While this material may not be ideal for comprising the entire stent, there is potential for use in combination with other materials. Furthermore, the ability to control degradation rates postimplantation opens new possibilities for biodegradable stents; additional magnetoelastic materials should be investigated for use in stenting applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.
... The formation of microcalcification is believed to originate from the apoptosis and differentiation of smooth muscle cells. 29,30 The negative correlation between 18 F-NaF uptake and α-SMA staining also proved this relationship. Although the mechanism is still unknown, the calcified nodules in these carotid plaques are considered a consequence of smooth muscle cell death. ...
Backgroud:
Vascular calcification is currently recognized as an important pathobiological process in atherosclerosis, but the mechanism remains elusive. Given the similarities in vascular calcification and bone formation,18F-sodium fluoride (18F-NaF) is now considered a novel marker of vascular calcification. This study aimed to correlate18F-NaF accumulation with the histological characterization of vascular calcification in carotid plaques.
Methods:
A total of 8 patients who were undergoing carotid endarterectomy (CEA) for carotid artery stenosis were recruited. Before CEA,18F-NaF positron emission tomography and computed tomography (PET-CT) studies were conducted.18F-NaF uptake was measured by the maximum standardized uptake value and the target-to-background ratio. The Hounsfield unit (HU) value was also measured. Postoperative carotid plaques were investigated by hematoxylin and eosin staining, alizarin red staining, and immunohistochemistry (alpha-smooth muscle actin and CD68).
Results:
18
F-NaF uptake was observed in the bilateral carotid bifurcation of all patients. Compared with the pathology results, there was a significant correlation between tracer activity in the carotid plaques and the calcification in the corresponding histological sections (integrated optical density [IOD]: r = .781, P = .022; positive area: r = .765, P = .027). A negative correlation was observed between18F-NaF uptake and smooth muscle cell staining (IOD: r = -.710, P = .049).18F-NaF uptake did not correlate with carotid artery stenosis, HU value, or inflammation.
Conclusions:
18
F-NaF PET-CT is a noninvasive imaging method for the assessment of calcification in human carotid atherosclerotic plaques and a promising approach to studying calcification in atherosclerotic lesions.
... 15 Of these subunits, IKKb is the essential kinase that mediates IjB phosphorylation. 15 In this study, we produced mice with a deletion of IKKb in vascular smooth muscle cells (VSMCs), a cell type contributing to calcification, 16 and subjected them to CaCl 2 -mediated aortic injury, which is an established animal model for vascular calcification. 17,18 Unexpectedly, we observed more calcification in the aorta of IKKb-deleted mice and found the activation of b-catenin and Runt-related transcription factor 2 (Runx2) as a possible mechanism. ...
Background:
Vascular calcification was previously considered as an advanced phase of atherosclerosis; however, recent studies have indicated that such calcification can appear in different situations. Nevertheless, there has been a lack of mechanistic insight to explain the difference. For example, the roles of nuclear factor-κB, a major regulator of inflammation, in vascular calcification are poorly explored, although its roles in atherosclerosis were well documented. Herein, we investigated the roles of nuclear factor-κB signaling in vascular calcification.
Methods and results:
We produced mice with deletion of IKKβ, an essential kinase for nuclear factor-κB activation, in vascular smooth muscle cells (VSMCs; KO mice) and subjected them to the CaCl2-induced aorta injury model. Unexpectedly, KO mice showed more calcification of the aorta than their wild-type littermates, despite the former's suppressed nuclear factor-κB activity. Cultured VSMCs from the aorta of KO mice also showed significant calcification in vitro. In the molecular analysis, we found that Runt-related transcription factor 2, a transcriptional factor accelerating bone formation, was upregulated in cultured VSMCs from KO mice, and its regulator β-catenin was more activated with suppressed ubiquitination in KO VSMCs. Furthermore, we examined VSMCs from mice in which kinase-active or kinase-dead IKKβ was overexpressed in VSMCs. We found that kinase-independent function of IKKβ is involved in suppression of calcification via inactivation of β-catenin, which leads to suppression of Runt-related transcription factor 2 and osteoblast marker genes.
Conclusions:
IKKβ negatively regulates VSMC calcification through β-catenin-Runt-related transcription factor 2 signaling, which revealed a novel function of IKKβ on vascular calcification.
... Several hypotheses involving cell-based and/or physical chemistry-based mineralization have been proposed to describe RP formations and the process of calcium-based stone formation and the association of the stone with RP [7,8,12]. Among these hypotheses, it has been suggested that the renal epithelial cells of uriniferous tubules [8,12], similar to the differentiation of pericytes [13], smooth muscle cells [14], and endothelial cells [15] can also differentiate towards osteoblast-like cells prompting vascular mineralization. Several noncollagenous proteins (NCPs) such as osteopontin (OPN), osteocalcin (OC), and bone sialoprotein (BSP) identified in bone extracellular matrix have been identified in renal calculi and are synthesized by renal epithelial tubular cells [10,12,16]. ...
Limited information exists on the anatomically-specific early stage events leading to clinically detectable mineral aggregates in the renal papilla. In this study, quantitative multiscale correlative maps of structural, elemental and biochemical properties of whole medullo-papillary complexes from human kidneys were developed. Correlative maps of properties specific to the uriniferous and vascular tubules using high-resolution X-ray computed tomography, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, and immunolocalization of noncollagenous proteins (NCPs) along with their association with anatomy specific biominerals were obtained. Results illustrated that intratubular spherical aggregates primarily form at the proximal regions distant from the papillary tip while interstitial spherical and fibrillar aggregates are distally located near the papillary tip. Biominerals at the papillary tip were closely localized with 10 to 50 μm diameter vasa recta immunolocalized for CD31 inside the medullo-papillary complex. Abundant NCPs known to regulate bone mineralization were localized within nanoparticles, forming early pathologic mineralized regions of the complex. Based on the physical association between vascular and urothelial tubules, results from light and electron microscopy techniques suggested that these NCPs could be delivered from vasculature to prompt calcification of the interstitial regions or they might be synthesized from local vascular smooth muscle cells after transdifferentiation into osteoblast-like phenotypes. In addition, results provided insights into the plausible temporal events that link the anatomically specific intratubular mineral aggregates with the interstitial biomineralization processes within the functional unit of the kidney.
... Breast arterial calcification (BAC) is a type of medial artery calcification that can be seen incidentally on mammography, and deposition of calcification in the arterial media is known as arteriosclerosis which is also known as Monckeberg's arteriosclerosis [1,2]. The deposition of calcified and non-calcified plaques in the arterial intima is known as atherosclerosis [3]. However, some recent studies suggested that Monckeberg's arteriosclerosis involves both the media and internal elastic lamina [4]. ...
Background
The aim of this study was to determine if breast arterial calcification (BAC) on mammography predicts myocardial ischemia (MI) on stress myocardial perfusion single-photon emission computed tomography (MPS). BAC is a type of medial artery calcification that can be seen incidentally on mammography, but the relationship between coronary artery calcification and MI on MPS is yet unknown.
Methods
A total of 435 consecutive women underwent mammography and stress MPS within 1 year of each other. BAC was quantitatively evaluated (0 - 13). Patients with known coronary artery diseases (CADs) such as coronary artery bypass grafting (CABG), percutaneous coronary intervention (PCI), MI, positive coronary angiogram or positive MPS were excluded from the study. Risk factors for CAD were obtained from a chart review.
Results
The mean age was 58 ± 8 years. BAC was found in 258 (59%) of the study population. BAC-positive patients were significantly older than BAC-negative patients (P < 0.0001), there were strong associations between BAC and hypertension (P = 0.0309), chronic kidney disease (CKD) (P = 0.0001), and diabetes (P = 0.0309), but there were significant associations between BACV and hyperlipidemia, family history of CAD, and smoking (P = 0.6856, P = 0.9642, and P = 0.087, respectively). The mean score of BAC was 5 ± 5 in patients with normal MPS and was 6 ± 6 in patients with abnormal MPS. There were no associations between total BAC and MPS results (P = 0.2095), and between BAC categories and MPS result (P = 0.3069).
Conclusions
Based on our study, the presence and severity of BAC on screening or diagnostic mammography do not predict MI on stress MPS, and further cardiac workup based on the presence of BAC is not warranted. BAC is very common in mammography up to 59% and associated with age, diabetes, CKD, and hypertension. In contrast, the prevalence of MI is only 13% in women with BAC and associated with age, diabetes, CKD, hyperlipidemia, and impaired left ventricular function.
Diabetes is a major risk factor for cardiovascular disease. However, the exact mechanism by which diabetes contributes to vascular damage is not fully understood. The aim of this study was to investigate the role of SUMO-1 mediated SERCA2a SUMOylation in the development of atherosclerotic vascular injury associated with diabetes mellitus. ApoE−/− mice were treated with streptozotocin (STZ) injection combined with high-fat feeding to simulate diabetic atherosclerosis and vascular injury. Human aortic vascular smooth muscle cells (HAVSMCs) were treated with high glucose (HG, 33.3 mM) and palmitic acid (PA, 200 µM) for 24 h to mimic a model of diabetes-induced vascular injury in vitro. Aortic vascular function, phenotypic conversion, migration, proliferation, intracellular Ca²⁺ concentration, the levels of small ubiquitin-like modifier type 1 (SUMO1), SERCA2a and SUMOylated SERCA2a were detected. Diabetes-induced atherosclerotic mice presented obvious atherosclerotic plaques and vascular injury, companied by significantly lower levels of SUMO1 and SERCA2a in aorta. HG and PA treatment in HAVSMCs reduced the expressions of SUMO1, SERCA2a and SUMOylated SERCA2a, facilitated the HAVSMCs phenotypic transformation, proliferation and migration, attenuated the Ca²⁺ transport, and increased the resting intracellular Ca²⁺ concentration. We also confirmed that SUMO1 directly bound to SERCA2a in HAVSMCs. Overexpression of SUMO1 restored the function and phenotypic contractile ability of HAVSMCs by upregulating SERCA2a SUMOylation, thereby alleviating HG and PA-induced vascular injury. These observations suggest an essential role of SUMO1 to protect diabetes-induced atherosclerosis and aortic vascular injury by the regulation of SERCA2a-SUMOylation and calcium homeostasis.
Graphic abstract
Background:
Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system.
Methods:
To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide.
Results:
Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs.
Conclusion:
These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.
Soft tissue mineralization has been found to be a major component of diseases such as aortic valve stenosis and rheumatic heart disease. Cardiovascular mineralization has been suggested to follow mechanisms similar to those of bone formation with several cell culture models been developed over the years to provide mechanistic insights. These cell models have been characterized by a wide range of biochemical and molecular methods, which identified the presence of osteogenic markers and bone-like cells. However, there is a surprisingly small number of studies where the mineral formed in these cell culture models has been characterized by physico-chemical methods, and even fewer studies have compared this mineral to the one produced by bone cells in cultures. Here we investigated the morphology and composition of the minerals formed in cell cultures of vascular smooth muscle cells and bone cells. Electron microscopy and traditional cell mineralization assays were applied, revealing that vascular cells are indeed able to form calcified nodules of elemental composition similar to bone, however with different morphology. Comparison of morphologies of the two minerals to that found in cardiovascular tissue shows that some of tissue calcification resembles the calcified fibers produced by bone cells in vitro . These results suggest that the characterization of the mineral is of utmost importance and its morphology and chemical properties can contribute an important piece of information in the comprehensive analysis of soft tissue mineralization mechanisms, both in in vitro cell culture as well as in clinical samples.
Periodontitis is a disease caused by infection and immunological imbalance, which often leads to the destruction of periodontal tissue. Programmed death protein 1 (PD-1) and its ligand: programmed death ligand 1 (PD-L1) are important “immune checkpoint” proteins that have a negative regulatory effect on T cells and are targets of immunotherapy. Studies have shown that the expression of PD-1 and PD-L1 in patients with periodontitis is higher than that in healthy individuals. The keystone pathogen Porphyromonas gingivalis (P. gingivalis) is believed to be the main factor driving the upregulation of PD-1/PD-L1. High expression of PD-1/PD-L1 can inhibit the inflammatory response and reduce the destruction of periodontal supporting tissues, but conversely, it can promote the “immune escape” of P. gingivalis, thus magnifying infections. In addition, the PD-1/PD-L1 pathway is also associated with various diseases, such as cancer and Alzheimer's disease. In this review, we discuss the influence and mechanism of the PD-1/PD-L1 pathway as a “double-edged sword” affecting the occurrence and development of periodontitis, as well as its function in periodontitis-related systemic disorders. The PD-1/PD-L1 pathway could be a new avenue for periodontal and its related systemic disorders therapy.
Atherosclerosis (AS), leading to gradual occlusion of the arterial lumen, refers to the accumulation of lipids and inflammatory debris in the arterial wall. Despite therapeutic advances over past decades including intervention or surgery, atherosclerosis is still the most common cause of cardiovascular diseases and the main mechanism of death and disability worldwide. Vascular smooth muscle cells (VSMCs) play an imperative role in the occurrence of atherosclerosis and throughout the whole stages. In the past, there was a lack of comprehensive understanding of VSMCs, but the development of identification technology, including in vivo single-cell sequencing technology and lineage tracing with the CreERT2-loxP system, suggests that VSMCs have remarkable plasticity and reevaluates well-established concepts about the contribution of VSMCs. Transcription factors, a kind of protein molecule that specifically recognizes and binds DNA upstream promoter regions or distal enhancer DNA elements, play a key role in the transcription initiation of the coding genes and are necessary for RNA polymerase to bind gene promoters. In this review, we highlight that, except for environmental factors, VSMC genes are transcriptionally regulated through complex interactions of multiple conserved cis-regulatory elements and transcription factors. In addition, through a series of transcription-related regulatory processes, VSMCs could undergo phenotypic transformation, proliferation, migration, calcification and apoptosis. Finally, enhancing or inhibiting transcription factors can regulate the development of atherosclerotic lesions, and the downstream molecular mechanism of transcriptional regulation has also been widely studied.
Atherosclerosis leads to heart attacks due to the formation of atherosclerotic plaques which narrow the arterial lumen, blocking the blood flow to the heart. These plaques are formed due to free radical-mediated oxidation of low-density lipoproteins (LDL) leading to their uncontrolled uptake by macrophages which in turn leads to inflammation. This inflammation and presence of oxidized LDL results in the proliferation and migration of macrophage and vascular smooth muscle cells leading to the formation and growth of atherosclerotic plaque which thereby lead to blockage of blood flow to heart. Plant phenolic compounds (the most abundantly occurring antioxidants in foods of plant origin) have been shown to inhibit the oxidation of low-density lipoproteins, which is considered a key mechanism in endothelial lesions occurring in atherosclerosis. These compounds have been shown to possess antioxidant capacity higher than that of ascorbic acid and α-tocopherol. In this review, the structural features of phenolic compounds (type of compound, degree of methoxylation, and the number of hydroxyl groups, etc.) that contribute to the antioxidant properties of phenolic compounds have been shown. Further, the chemical mechanism by which these features help in combating every step in free radical-mediated atherosclerotic plaque progression has been described. This review also sheds light on how the antioxidant properties of individual phenolic compounds lead to the inhibition of molecular pathways involved in inflammation leading to endothelial dysfunction.
Background
Increasing evidence has spurred a considerable evolution of concepts related to atherosclerosis, prompting the need to provide a comprehensive view of the growing literature. By retrieving publications in the Web of Science Core Collection (WoSCC) of Clarivate Analytics, we conducted a bibliometric analysis of the scientific literature on atherosclerosis to describe the research landscape.
Methods
A search was conducted of the WoSCC for articles and reviews serving exclusively as a source of information on atherosclerosis published between 2012 and 2022. Microsoft Excel 2019 was used to chart the annual productivity of research relevant to atherosclerosis. Through CiteSpace and VOSviewer, the most prolific countries or regions, authors, journals, and resource-, intellectual-, and knowledge-sharing in atherosclerosis research, as well as co-citation analysis of references and keywords, were analyzed.
Results
A total of 20,014 publications were retrieved. In terms of publications, the United States remains the most productive country (6,390, 31,93%). The most publications have been contributed by Johns Hopkins Univ (730, 3.65%). ALVARO ALONSO produced the most published works (171, 0.85%). With a betweenness centrality of 0.17, ERIN D MICHOS was the most influential author. The most prolific journal was identified as Atherosclerosis (893, 4.46%). Circulation received the most co-citations (14,939, 2.79%). Keywords with the ongoing strong citation bursts were “nucleotide-binding oligomerization (NOD), Leucine-rich repeat (LRR)-containing protein (NLRP3) inflammasome,” “short-chain fatty acids (SCFAs),” “exosome,” and “homeostasis,” etc.
Conclusion
The research on atherosclerosis is driven mostly by North America and Europe. Intensive research has focused on the link between inflammation and atherosclerosis, as well as its complications. Specifically, the NLRP3 inflammasome, interleukin-1β, gut microbiota and SCFAs, exosome, long non-coding RNAs, autophagy, and cellular senescence were described to be hot issues in the field.
Vascular calcification is an independent risk factor for acute cardiovascular events and a predictor of adverse prognosis; the abnormal fusion and degradation of autophagosomes and lysosomes are closely related to the calcification of VSMC and aortic AS plaque in ApoE−/− mice. Rab7 is a member of the Ras protein family and acts as a molecular switch in the fusion between autophagosomes and lysosomes. In this study, we found that the activation of the CD137-CD137L signal promoted calcification by inhibiting the expression and activity of Rab7, which regulates the degradation of autophagic cargo in vascular smooth muscle cells (VSMCs) and aortic atherosclerosis (AS) plaques in ApoE−/− mice. Knockdown of Rab7 impaired its tethering with the downstream molecule FYVE and coiled-coil containing 1 (FYCO1), which transports autophagosomes to lysosomes through microtubule motor kinesins and fuses with lysosomes to degrade the autophagic content. Overexpression of Rab7-alleviated calcification caused by the activation of the CD137 signaling pathway. In addition, FYCO1 knockdown promoted calcification even though the expression and activity of Rab7 were normal. Our results suggest that Rab7 is the target of CD137 signaling; Rab7 cannot interact with its downstream molecule FYCO1 when its activity and expression were inhibited by the activation of CD137 signaling pathway, thus inhibiting the autophagic degradation and promoting calcification.
Graphical abstract
Pathological mineralization (PTM) often occurs under inflammation and affects the prognosis of diseases, such as atherosclerosis and cancers. However, how the PTM impacts inflammation has not been well explored. Herein, poly lactic‐co‐glycolic acid (PLGA)/gelatin/hydroxyapatite (HA) electrospun nanofibers are rationally designed as an ideal PTM microenvironment biomimetic system for unraveling the role of PTM on inflammation. The results demonstrate that the inflammatory response decreases continuously during the process of mineralization. When mature macromineralization forms, the inflammation almost completely disappears. Mechanistically, the PTM formation is mediated by matrix proteins, local high calcium, and cell debris (nuclei), or actively regulated by the lysosomal/plasma membrane components secreted by macrophages. These inflammatory inducible factors (calcium, cell debris, etc.) can be “buried” through PTM process, resulting in reduced immune responses. Overall, the present study demonstrates that PTM is an innate mechanism of inflammation subsidence, providing valuable insight into understanding the action of mineralization on inflammation.
Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA-associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA-based analyses uncover depleted cardiac-specific processes, among other relevant functions, that are undetected by CA. Twenty BA-related microRNAs are identified, and two of them highly heart-enriched that are present in plasma. We describe a microRNA-gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research.
Biomineralization of skeletal components (e.g., bone and teeth) is generally accepted to occur under strict cellular regulation, leading to mineral–organic composites with hierarchical structures and properties optimized for their designated function. Such cellular regulation includes promoting mineralization at desired sites as well as inhibiting mineralization in soft tissues and other undesirable locations. In contrast, pathological mineralization, with potentially harmful health effects, can occur as a result of tissue or metabolic abnormalities, disease, or implantation of certain biomaterials. This progress report defines mineralization pathway components and identifies the commonalities (and differences) between physiological (e.g., bone remodeling) and pathological calcification formation pathways, based, in part, upon the extent of cellular control within the system. These concepts are discussed in representative examples of calcium phosphate‐based pathological mineralization in cancer (breast, thyroid, ovarian, and meningioma) and in cardiovascular disease. In‐depth mechanistic understanding of pathological mineralization requires utilizing state‐of‐the‐art materials science imaging and characterization techniques, focusing not only on the final deposits, but also on the earlier stages of crystal nucleation, growth, and aggregation. Such mechanistic understanding will further enable the use of pathological calcifications in diagnosis and prognosis, as well as possibly provide insights into preventative treatments for detrimental mineralization in disease.
Aims
Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells.
Materials and methods
Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively.
Key findings
Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression.
Significance
In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis.
Chemical compounds studied in this study
HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).
Objective
To explore the potential mechanism of KMUP-1 in the vascular calcification of chronic renal failure (CRF) through mediating NO/cGMP/PKG pathway, and provide novel insights into the CRF treatment.
Methods
CRF rats were treated by KMUP-1 with/without L-NNA (a NOS inhibitor) and then performed by ELISA, alizarin red staining, Von Kossa staining, Masson's trichrome, Sirius red staining and CD3 immunohistochemical staining. Simultaneously, vascular smooth muscle cells (VSMCs) were collected from rats to confirm the effect of KMUP-1 on vascular calcification in vitro via NO/cGMP/PKG pathway. Besides, protein and mRNA expressions were determined via Western blotting and qRT-PCR, respectively.
Results
CRF rats were elevated in 24-h urine protein, blood urea nitrogen (BUN), serum creatinine, Cys-C levels and inflammatory cytokines. Besides, CRF rats also showed increased calcium content and ALP level with up-regulated mRNA of osteogenic differentiation-related markers. Furthermore, the up-regulated expressions of eNOS and PKG, as well as down-regulated levels of NOx and cGMP were also found in CRF rats. However, renal failure and vascular calcification of CRF were improved significantly by KMUP-1 treatment via activation of NO/cGMP/PKG pathway. Moreover, KMUP-1 treatment attenuated calcified VSMCs, accompanied by the decreases in the calcified nodules, level of calcium and activity of ALP. In addition, either L-NNA treatment for CRF rats or the calcified VSMCs could antagonize the improving effect of KMUP-1.
Conclusion
KMUP-1 can improve the renal function and vascular calcification in CRF rats at least in part by activating NO/cGMP/PKG pathway.
In 2018, cardiovascular disease (CVD) was the leading cause of death among women, and current CVD prevention paradigms may not be sufficient in this group. In that context, it has recently been proposed that detection of calcification in breast arteries may help improve CVD risk screening and assessment in apparently healthy women. This review provides an overview of breast arterial anatomy; and the epidemiology, pathophysiology, and measurement of breast artery calcium (BAC); and discusses the features of the BAC-CVD link. The potential clinical applications that BAC may offer for CVD prevention in the context of current clinical practice guidelines and recommendations are also discussed. Finally, current gaps in evidence gaps are outlined, and future directions in the field are explored with a focus on the implementation of BAC mammography as a CVD risk-screening tool in routine clinical practice.
Thrombotic disorders increase the risk of cardiovascular/cerebrovascular complications and represent a major health problem worldwide. Anticoagulants are extensively used in treatment of these disorders. Vitamin K antagonists, like Warfarin, are frequently used in medication. Vascular calcification (VC) is a significant side-effect of vitamin K antagonists especially Warfarin. There is an urgent need to find natural, efficient, non-toxic, and cost effective anticoagulants without dangerous side-effect like VC. In the present study, we evaluated the potential of thirteen fermentation broth extracts of actinobacteria (FBEA) (200 µg mL ⁻¹ ) to prolong whole blood prothrombin time (PT)/international normalized ratio (INR) and activated partial thromboplastin time (APTT). The fractions of the most effective FBEA were further investigated for their inhibitory effect on VC. The results showed PT/INR of the healthy blood samples was sensitive to the presence of five FBEA. Their INR index fell in the 1.2 to 8.6 range and six FBEA prolonged both PT/INR and APTT parameters (1.7-5 INR, and 46-59 s for APTT). The fractions of Kribbella sp. UTMC 267 FBE (200 µg mL ⁻¹ ), as the most efficient FBE, only inhibited intrinsic and common pathways of coagulation (APTT). Under calcification condition, Kribbella sp. UTMC 267 fractions (20 µg mL ⁻¹ ) showed significant inhibitory effect on VC in alizarin red staining (13.3-76 %) and alkaline phosphatase activity of VSMCs (33-62%). They also inhibited osteopontin mRNA expression in treated vascular smooth muscle cells (VSMCs) under that situation. So, we can introduce Kribbella sp. UTMC 267 FBE as a good candidate for more investigation on thrombotic medication. © 2019, Iranian Journal of Pharmaceutical Research. All rights reserved.
Traditionally in the field of kidney transplantation, major attention has been focused on acute rejection and opportunistic infections. Nowadays, these factors are no longer a major problem and many patients are living with a functioning kidney graft for many years. Therefore, the attention has been shifted to effective surveillance of these patients.
The kidney plays a central role in the physiology of the body. Therefore, patients on the waiting list with renal failure have major abnormalities in, for example, the cardiovascular system, bone metabolism, and body composition.
After transplantation, these abnormalities remain unaltered or might regress partially or completely. The picture becomes even more complex because immunosuppressive agents have many systemic side effects.
In this book, some of these complex processes are described by authors who are recognized experts in their field. After reading this important information, it will become clear that the care of renal transplant patients, for various reasons, is a complex and highly specialized field.
Johannes P. van Hooff Department of Internal Medicine division of Nephrology Maastricht University Medical Center Maastricht, The Netherlands
Cellular senescence is the permanent cell cycle arrest induced either by chronological ageing or extrinsic stimuli. Recent researches have identified cellular senescence as an important mechanism for atherosclerosis, which is the essential pathophysiological contributor to cardiovascular diseases (CVDs). The sirtuins are a family of cellular deacetylases with fundamental abilities to regulate cellular metabolism and a variety of physiological activities. Previous studies have revealed the anti-ageing functions of sirtuins as the longevity-associated proteins. These advances indicate the potential beneficial functions of sirtuins in atherosclerosis by affecting cellular senescence. Herein, we review the recent findings about sirtuins in regulating atherosclerotic cellular senescence, and discuss the possibility of activating sirtuins as a therapeutic strategy for combating atherosclerosis.
It is a rare case of phlebolith
Vascular smooth muscle cells (VSMCs) transdifferentiate into osteoblast-like cells during vascular calcification, inducing active remodeling and calcification of the extracellular matrix (ECM). Intracellular and extracellular enzymes, such as lysyl hydroxylase 1 (PLOD1) and lysyl oxidase (LOX), contribute to ECM maturation and stabilization. We assessed the contribution of these enzymes to hyperphosphatemia (HPM)-induced calcification. Human and murine VSMCs were differentiated into functional osteoblast-like cells by HPM conditioning. HPM promoted ECM calcification and up-regulated osteoblast markers associated with induction of LOX and PLOD1 expression and with an increase in ECM-insoluble collagen deposition. Murine VSMCs from transgenic mice overexpressing LOX (TgLOX) exhibited an increase in HPM-dependent calcification and osteoblast commitment compared with wild-type cells. Similarly, enhanced HPM-induced calcification was detected in aorta from TgLOX. Conversely, β-aminopropionitrile (a LOX inhibitor) and LOX knockdown abrogated VSMC calcification and transdifferentiation. We found a significant positive association between LOX expression and vascular calcification in human atherosclerotic lesions. Likewise, 2,2'-dipyridil (a PLOD inhibitor) and PLOD1 knockdown impaired HPM-induced ECM mineralization and osteoblast commitment. Our findings identify LOX and PLOD as critical players in vascular calcification and highlight the importance of ECM remodeling in this process.-Jover, E., Silvente, A., Marín, F., Martínez-González, J., Orriols, M., Martinez, C. M., Puche, C. M., Valdés, M., Rodriguez, C., Hernández-Romero, D. Inhibition of enzymes involved in collagen cross-linking reduces vascular smooth muscle cell calcification.
Previous studies in our laboratory demonstrated messenger RNA for bone morphogenetic protein-2a in human calcified plaque, suggesting that arterial calcification is a regulated process, similar to osteogenesis. To further test this hypothesis, we have isolated and cloned a subpopulation of cells from bovine aortic media that show osteoblastic potential. These novel cells are primarily distinguished from smooth muscle cells by expression of a surface marker preliminarily identified as a modified form of the ganglioside sialyl-lactosylceramide (GM3). Osteoblastic potential was indicated by high levels of alkaline phosphatase and collagen I, expression of osteopontin and osteonectin (SPARC), and production of bone-specific osteocalcin and hydroxyapatite. Cultures of these cells were stimulated to form increased numbers of calcium-mineral-producing nodules by the oxysterol 25-hydroxycholesterol as well as by transforming growth factor-beta 1, both known to be present in atherosclerotic lesions. The stimulation of calcifying vascular cells in the artery wall by these two factors suggests a possible mechanism for the colocalization of calcification with atherosclerosis in vivo.
In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.
Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.
To assess the size of the lipid pool and the number of smooth muscle cells and monocyte/macrophages in human aortic plaques that were intact and to compare the results with those in aortic plaques undergoing ulceration and thrombosis.
The lipid pool was measured as a percentage of the total cross sectional area of the plaque. Immunohistochemistry was used to identify cell types (monocytes/macrophages (M phi) by EBM11 and HAM56, smooth muscle cells by alpha actin). The area of the tissue occupied by each cell type was measured by quantitative microscopy in the peripheral (shoulder) area of the plaque and the plaque cap. Absolute counts of each cell type were expressed as the ratio of SMC:M phi.
Aortas were obtained at necropsy from men aged less than 69 years who died suddenly (within 6 hours of the onset of symptoms) of ischaemic heart disease. 155 plaques from 13 aortas were studied. Four aortas showed intact plaques only (group A, n = 31). Nine aortas showed both intact plaques (group B, n = 79) and plaques that were undergoing thrombosis (group C, n = 45).
In 41 (91.1%) of the 45 plaques undergoing thrombosis (group C) lipid pools occupied more than 40% of the cross sectional area of the plaque. Only 12 (10.9%) of the 110 intact plaques (groups A + B) had lipid pools of this size. The mean size of the lipid pool in plaques of groups A, B, and C was 12.7%, 27.3% and 56.7% respectively. Compared with intact plaques those undergoing thrombosis contained a smaller volume of smooth muscle cells (2.8% v 11.8%) and a larger volume of monocyte/macrophages (13.7% v 2.9%) in the plaque cap. The ratio of the number of smooth muscle cells to monocytes/macrophages was 7.8 in group A plaques, 4.1 in group B plaques, and 1.0 in group C plaques. This gradient was the result of an absolute increase in monocyte/macrophages and an absolute decrease in smooth muscle cells.
In the aorta ulceration and thrombosis were characteristic of plaques with a high proportion of their volume occupied by extracellular lipid, and in which there was a shift toward a preponderance of monocyte/macrophages compared with smooth muscle cells in the cap.
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.
Objective — Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis.
Methods and Results — By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a “hill-and-valley” growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-β2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection.
Conclusions — Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.
Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of
Background:
Lipids play a significant role in the process of calcification of bioprostheses. We assessed whether lipid extraction by ethanol, ether, or a surfactant could mitigate calcification of glutaraldehyde-treated bioprostheses.
Methods:
On 200 bovine pericardium samples pretreated with 0.6% glutaraldehyde, lipid extraction was carried out by ethanol, ether, or the tween 80 surfactant, and combinations thereof. The treated tissues were implanted subcutaneously in 50 juvenile rats for 4 and 6 months. Lipids were analyzed by Fourier transform infrared spectrophotometer and chromatography before implantation. Calcium content of implanted tissues was assessed by atomic absorption spectrometer.
Results:
Ethanol, ether, or surfactant did mitigate calcification. The most efficient pretreatments were the combination of ethanol and surfactant (calcium content: 15.5+/-6.8 microg/mg dry tissue after 6 months implantation) or the combination of ethanol, ether, and surfactant (13.1+/-6.2 microg/mg dry tissue) when compared with surfactant alone (42.9+/-12.7 microg/mg dry tissue).
Conclusions:
Ethanol or the combination of ethanol and ether added to the currently used glutaraldehyde-surfactant treatment further mitigates calcification.
The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa's method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers alpha-SM actin, SM22alpha, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA. In contrast, OP mRNA was barely detectable in calcified human VSMCs and pericyte nodules, nor was OP protein detected, suggesting that OP was not necessary for calcification to occur. These studies reveal that human VSMCs are capable of inducing calcification and that MGP may have a role in human vascular calcification.
To help determine if coronary angiography can predict the site of a future coronary occlusion that will produce a myocardial infarction, the coronary angiograms of 42 consecutive patients who had undergone coronary angiography both before and up to a month after suffering an acute myocardial infarction were evaluated. Twenty-nine patients had a newly occluded coronary artery. Twenty-five of these 29 patients had at least one artery with a greater than 50% stenosis on the initial angiogram. However, in 19 of 29 (66%) patients, the artery that subsequently occluded had less than a 50% stenosis on the first angiogram, and in 28 of 29 (97%), the stenosis was less than 70%. In every patient, at least some irregularity of the coronary wall was present on the first angiogram at the site of the subsequent coronary obstruction. In only 10 of the 29 (34%) did the infarction occur due to occlusion of the artery that previously contained the most severe stenosis. Furthermore, no correlation existed between the severity of the initial coronary stenosis and the time from the first catheterization until the infarction (r2 = 0.0005, p = NS). These data suggest that assessment of the angiographic severity of coronary stenosis may be inadequate to accurately predict the time or location of a subsequent coronary occlusion that will produce a myocardial infarction.
CARDIOVASCULAR disease remains the chief cause of death in the United States and Western Europe, and atherosclerosis, the principal cause of myocardial and cerebral infarction, accounts for the majority of these deaths.1 This review, like its predecessor,2 will not attempt to cover all literature on atherosclerosis. In a previous review of the pathogenesis of atherosclerosis,2 Glomset and I discussed various hypotheses of atherogenesis2 3 4 5 6 7 and emphasized the importance of intimal smooth-muscle proliferation as the key event in the development of the advanced lesions of atherosclerosis. The response-to-injury hypothesis of atherogenesis2 3 4 5 6 proposes that "injury" to the endothelium is the initiating event in . . .
Smooth muscle proliferation has been recognized as central to the pathology of both major forms of vascular disease: atherosclerosis and hypertension. Recent advances in our knowledge of mechanisms of control of proliferation suggest that events occurring in adult animals may recapitulate portions of the developmental biology of the smooth muscle cell. This review attempts to consider the current state of knowledge of the mechanisms controlling smooth muscle proliferation in these two diseases, to put that knowledge into the context of what is known about smooth muscle biology, and to offer two hypotheses on the possible roles of smooth muscle developmental biology in manifestations of atherosclerosis and hypertension in adult humans.
Whether human coronary arteries undergo compensatory enlargement in the presence of coronary disease has not been clarified. We studied histologic sections of the left main coronary artery in 136 hearts obtained at autopsy to determine whether atherosclerotic human coronary arteries enlarge in relation to plaque (lesion) area and to assess whether such enlargement preserves the cross-sectional area of the lumen. The area circumscribed by the internal elastic lamina (internal elastic lamina area) was taken as a measure of the area of the arterial lumen if no plaque had been present. The internal elastic lamina area correlated directly with the area of the lesion (r = 0.44, P less than 0.001), suggesting that coronary arteries enlarge as lesion area increases. Regression analysis yielded the following equation: Internal elastic lamina area = 9.26 + 0.88 (lesion area) + 0.026 (age) + 0.005 (heart weight). The correlation coefficient for the lesion area was significant (P less than 0.001), whereas the correlation coefficients for age and heart weight were not. The lumen area did not decrease in relation to the percentage of stenosis (lesion area/internal elastic lamina area X 100) for values between zero and 40 percent but did diminish markedly and in close relation to the percentage of stenosis for values above 40 percent (r = -0.73, P less than 0.001). We conclude that human coronary arteries enlarge in relation to plaque area and that functionally important lumen stenosis may be delayed until the lesion occupies 40 percent of the internal elastic lamina area. The preservation of a nearly normal lumen cross-sectional area despite the presence of a large plaque should be taken into account in evaluating atherosclerotic disease with use of coronary angiography.
New observations portray calcification processes as similar whether occurring normally or pathologically. Most forms of calcification are initiated by membranous organelles, ie, extracellular, calcifying "matrix vesicles" or intracellular mitochondria. Matrix vesicles promote calcification through calcium-binding phospholipids and phosphatase activity. Mitochondria use a forceful, inwardly directed Ca and phosphate transport mechanism. After mineral initiation, the proliferation of mineral crystals is dependent on regulatory factors, such as extracellular Ca2+ and PO4(3-) and other mineral inhibitors and promoters. Calcific diseases are defined as those in which (1) Ca uptake is early, (2) calcification is importantly related to dysfunction, and (3) the control of calcification may lead to decreased morbidity or enhanced diagnostic capability. Calcific diseases include such well-known entities as crystal deposition arthritis, atherosclerosis, calcific valvular sclerosis, tumor calcification, dental plaque, and dysfunctional calcification occurring in implanted cardiovascular devices.
Nearly 40 years after its invention, the angiogram is still considered by most physicians to be the "gold standard" for defining coronary anatomy. Careful investigations have revealed many deficiencies inherent in this approach. The purpose of this article is to outline the evidence that our current preoccupation with coronary "luminology" may be misguided and to propose a rational paradigm for future practice and investigation. Angiography depicts coronary anatomy from a planar two-dimensional silhouette of the lumen. Angiography is limited in resolution to four or five line pairs per millimeter. Confounding factors include vessel tortuosity, overlap of structures, and the effects of lumen shape. After intervention, a hazy, broadened silhouette may overestimate the actual gain in lumen size. Studies show marked disparity between the apparent severity of lesions and their physiological effects. After myocardial infarction, cardiologists too often do not make an attempt to demonstrate the physiological significance of the stenosis before performing percutaneous coronary revascularization. Similarly, the allure of a better, more gratifying angiogram with new interventional devices appears to be a dominant factor in their popularity. Interventional cardiologists should be aware that techniques yielding marked angiographic benefit may also generate important but unrecognized hazards. The dissociation between the angiogram and clinical outcome should influence future research efforts. Our review of the literature indicates that we may benefit from shifting the current focus and preoccupation with coronary luminology to achieving the desired clinical end point: promoting survival and long-term freedom from myocardial infarction and the disabling symptoms of coronary heart disease.
The vascular smooth muscle cell (SMC) in mature animals is a highly specialized cell whose principal function is contraction. The fully differentiated or mature SMC proliferates at an extremely low rate and is a cell almost completely geared for contraction. It expresses a unique repertoire of contractile proteins, ion channels, and signaling molecules that are required for its contractile function and that when taken in aggregate clearly distinguish it from any other cell type. During vasculogenesis, however, the SMC's principal function is proliferation and production of matrix components of the blood vessel wall. Moreover, even in mature animals, the SMC retains remarkable plasticity, such that it can undergo relatively rapid and reversible changes in its phenotype in response to changes in local environmental cues normally required for maintenance of its differentiated state. A key to understanding SMC differentiation is to identify the key environmental signals and factors that induce or maintain the differentiated state of the SMC and to determine the molecular mechanisms that control the coordinate expression of genes encoding for proteins that are necessary for the contractile function of the SMC. The purpose of this review is to summarize our current knowledge of the regulation of SMC differentiation, with a particular emphasis on consideration of how this process is controlled during normal vascular development and how these control processes might be altered in vascular diseases such as atherosclerosis, which are characterized by marked alterations in the differentiated state of the SMC.
Matrix vesicles are extracellular 100-nanometer-diameter membrane-invested particles selectively located within the matrix of bone, cartilage, and predentin. They serve as the initial site of calcification in all skeletal tissues. Matrix vesicle biogenesis occurs by polarized budding and pinching off of vesicles from specific regions of the outer plasma membrane of chondrocytes, osteoblasts, and odontoblasts. Seeding of selected areas of matrix with matrix vesicles explains the localized distribution of subsequent zones of mineralization. Matrix vesicle biogenesis in the growth plate is linked to the chondrocyte cell cycle and reflects a stage in programmed cell death (apoptosis). Generation of initial hydroxyapatite mineral crystals occurs within the matrix vesicle membrane during Phase 1 of biologic mineralization. Phase 1 is controlled by phosphatases (including alkaline phosphatase) and Ca-binding molecules with which the matrix vesicles are well endowed. Phase 2 of biologic mineralization begins with breakdown of matrix vesicle membranes, exposing preformed hydroxyapatite to the extracellular fluid after which mineral crystal proliferation is governed by extracellular conditions. Phase 1 and Phase 2 of mineralization are under cellular control. Phase 1 is initiated by cells generating calcifiable matrix vesicles and releasing them into sites of intended calcification. Phase 2 is controlled by cells regulating extracellular ionic conditions and matrix composition.
Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.
How an atherosclerotic plaque evolves from minimal diffuse intimal hyperplasia to a critical lesion is not well understood. Cellular proliferation is a relatively infrequent and modest event in both primary and restenotic coronary atherectomy specimens, leading us to believe that other processes, such as the formation of extracellular matrix, cell migration, neovascularization, and calcification might be more important for lesion formation. The investigation of proteins that are overexpressed in plaque compared with the normal vessel wall may provide clues that will help determine which of these processes are key to lesion pathogenesis. One such molecule, osteopontin (OPN), is an arginine-glycine-aspartate-containing acidic phosphoprotein recently shown to be a novel component of human atherosclerotic plaques and selectively expressed in the rat neointima following balloon angioplasty. Using in situ hybridization and immunohistochemical methods, we demonstrate that in addition to macrophages, smooth muscle and endothelial cells synthesize OPN mRNA and protein in human coronary atherosclerotic plaque specimens obtained by directional atherectomy. In contrast, OPN mRNA and protein were not detected in nondiseased vessel walls. Furthermore, extracellular OPN protein collocalized with sites of early calcification in the plaque that were identified with a sensitive modification of the von Kossa staining technique. These findings, combined with studies showing that OPN has adhesive, chemotactic, and calcium-binding properties, suggest that OPN may contribute to cellular accumulations and dystrophic calcification in atherosclerotic plaques.
Clones were derived by dilute plating from cultured aortic smooth muscle cells of 12-day-old rats. Clones Pup I to III resemble uncloned smooth muscle cultures from aortas of rat pups and from adult rat neointimas. They have a cobblestone morphology and proliferate in plasma-derived serum. By Northern analysis they contain platelet-derived growth factor B chain (PDGF-B) mRNA and high levels of CYPIA1, elastin, and osteopontin mRNAs, and they lack platelet-derived growth factor alpha-receptor (PDGF-alpha R) mRNA. In contrast, Pup V resembles smooth muscle cultures derived from uninjured adult rat arteries. It has an elongated morphology and proliferates poorly in plasma-derived serum. This clone expresses PDGF-alpha R mRNA, low levels of elastin and osteopontin mRNAs, and lacks CYPIA1 and PDGF-B mRNAs. Pup IV and VI have most of the properties of Pup I to III. We conclude that uncloned pup cultures are heterogeneous, but that the growth properties and gene expression pattern described for the uncloned culture is characteristic of individual clones within the population.
Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability.
The consensus of evidence from angiographic trials demonstrates both coronary artery and clinical benefits from lowering of lipids by a variety of regimens. The findings of reduced arterial disease progression and increased regression have been convincing but, at best, modest in their magnitude. For example, among those treated intensively in FATS, the mean improvement in proximal stenosis severity per patient was < 1% stenosis, and only 12% of all lesions showed convincing regression. In view of these modest arterial benefits, the associated reductions in cardiovascular events have been surprisingly great. For example, coronary events were reduced 75% in FATS; this was entirely a result of a 93% reduction in the likelihood that a mildly or moderately diseased arterial segment would experience substantial progression to a severe lesion at the time of a clinical event. We believe that the magnitude of the clinical benefit is best explained in terms of this observation, according to the following lines of reasoning. Clinical events most commonly spring from lesions that are initially of mild or moderate severity and then abruptly undergo a disruptive transformation to a severe culprit lesion. The process of plaque fissuring, leading to plaque disruption and thrombosis, triggers most clinical coronary events. Fissuring is predicted by a large accumulation of core lipid in the plaque and by a high density of lipid-laden macrophages in its thinned fibrous cap. Lesions with these characteristics constitute only 10-20% of the overall lesion population but account for 80-90% of the acute clinical events. In the experimental setting, normalization of an atherogenic lipid profile substantially decreases the number of lipid-laden intimal macrophages (foam cells) and depletes cholesterol from the core lipid pool. In the clinical setting, intensive lipid lowering virtually halts the progression of mild and moderate lesions to clinical events. Thus, the reduction in clinical events observed in these trials appears to be best explained by the relation of the lipid and foam cell content of the plaque to its likelihood of fissuring and by the effects of lipid-lowering therapy on these "high-risk" features of plaque morphology. The composite of data presented here supports the hypothesis that lipid-lowering therapy selectively depletes (regresses) that relatively small but dangerous subgroup of fatty lesions containing a large lipid core and dense clusters of intimal macrophages. By doing so, these lesions are effectively stabilized and clinical event rate is accordingly decreased.
Atherosclerotic calcification and osteoporosis often coexist in patients, yielding formation of bone mineral in vascular walls and its simultaneous loss from bone. To assess the potential role of lipoproteins in both processes, we examined the effects of minimally oxidized low-density lipoprotein (MM-LDL) and several other lipid oxidation products on calcifying vascular cells (CVCs) and bone-derived preosteoblasts MC3T3-E1. In CVCs, MM-LDL but not native LDL inhibited proliferation, caused a dose-dependent increase in alkaline phosphatase activity, which is a marker of osteoblastic differentiation, and induced the formation of extensive areas of calcification. Similar to MM-LDL, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and the isoprostane 8-iso prostaglandin E2 but not PAPC or isoprostane 8-iso prostaglandin F2 alpha induced alkaline phosphatase activity and differentiation of CVCs. In contrast, MM-LDL and the above oxidized lipids inhibited differentiation of the MC3T3-E1 bone cells, as evidenced by their stimulatory effect on proliferation and their inhibitory effect on the induction of alkaline phosphatase and calcium uptake. These results suggest that specific oxidized lipids may be the common factors underlying the pathogenesis of both atherosclerotic calcification and osteoporosis.
To determine the predictive value of coronary calcifications for coronary heart disease events in high-risk, asymptomatic adults:
A prospective cohort study of 1,461 high-risk, asymptomatic subjects were followed for 55 months with a 98% success rate. Coronary risk factor assessment and cardiac fluoroscopy with digital subtraction enhancement were performed to determine the number of calcified coronary arteries.
Fifty-eight percent of this cohort (852 subjects) had fluoroscopically detectable coronary calcification: 437 (30%) had calcium in one, 253 (17%) in two, and 162 (11%) in all three coronary vessels. There were 90 (6%) deaths, 35 (39%) attributable to coronary heart disease, and 43 (3%) nonfatal myocardial infarctions. Subjects with calcification in more than one major coronary artery were 2.2 times more likely to suffer coronary death or nonfatal infarction (P = 0.001) than were subjects with one or no calcified arteries. Multivariable logistic regression analysis showed that only the number of calcified arteries, age, total cholesterol, history of diabetes, and left ventricular hypertrophy by electrocardiogram were associated independently with the incidence of coronary death or infarction in these subjects.
Coronary calcification predicts coronary heart disease death or infarction in high-risk asymptomatic adults as well as do standard risk factors.
At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.
The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions.
Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles.
We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.
Thrombosis on the substrate of a disrupted plaque causes most acute coronary events. The physical integrity of the plaque thus governs the most important clinical manifestations of atherosclerosis. Of particular importance is the extracellular matrix of the fibrous capsule overlying the thrombogenic core of the atheroma.
Stable atheroma generally have thick fibrous caps, and smaller lipid cores than lesions that have ruptured. Accumulating evidence supports a key role for inflammation as another critical determinant of the stability of human atherosclerotic plaques.
Plaques that rupture usually have more abundant leucocytic infiltrates than those considered stable. Inflammatory mediators such as cytokines can influence several biological processes that regulate the stability of the plaque's fibrous cap, and thus its resistance to rupture.
For example, interferon-γ produced by activated T lymphocytes within atheroma inhibits the production of interstitial forms of collagen by human vascular smooth muscle cells. Inflammatory cytokines such as interleukin-1, tumour necrosis factor (TNF) and CD-40 ligand (a cell surface homologue of TNFα) can also elicit the expression by macrophages and smooth muscle cells of proteolytic enzymes that can weaken the extracellular matrix.
We have hypothesised that lipid lowering reduces stimuli for the inflammatory response within the complex atherosclerotic lesion. Recent studies in rabbits with experimentally produced atherosclerosis have indeed shown that lipid lowering can (i) reduce macrophage numbers, (ii) decrease expression of the collagenolytic enzyme MMP-1, and (iii) reinforce the plaque's fibrous skeleton by increasing the content of interstitial collagen.
By reducing local inflammation, lipid lowering can thus stabilise the plaque's fibrous cap, rendering the atheroma less prone to rupture and to precipitate thrombotic complications.
These observations provide a mechanistic basis for understanding the marked reduction in acute coronary events and cerebrovascular accidents observed in patients treated with agents that reduce plasma lipids.
A significant association between vascular calcification and osteoporosis has been noted, suggesting that calcium homeostasis is important in vascular calcification as well as in osteoporosis. Moreover, results of our previous studies suggest that calcium-regulating hormones such as parathyroid hormone-related peptide (PTHrP) may modulate vascular calcification. Therefore, we hypothesized that 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] may have a direct impact on the calcium-regulating system of vascular smooth muscle cells, resulting in deposition of calcium in vascular wall.
We investigated the effect of 1,25(OH)2D3 on in vitro calcification by bovine vascular smooth muscle cells (BVSMCs). 1,25(OH)2D3 dose dependently increased BVSMC calcification and alkaline phosphatase activity. 1,25(OH)2D3 also decreased secretion of PTHrP by BVSMCs in a dose-dependent manner and depressed its gene expression. Furthermore, exogenous PTHrP (fragment 1-34) antagonized the stimulatory effect of 1,25(OH)2D3 on BVSMCs. Finally, 1,25(OH)2D3 dose dependently increased the expression of the osteopontin gene, one of the bone matrix proteins in BVSMCs, contributing to its stimulatory action on BVSMC calcification.
These data suggest that 1,25(OH)2D3 exerts a stimulatory effect on vascular calcification through direct inhibition of the expression of PTHrP in BVSMCs as an endogenous inhibitor of vascular calcification. Moreover, the stimulatory effects of 1,25(OH)2D3 on alkaline phosphatase activity and osteopontin expression may contribute to its promoting action in vascular calcification.
The association between oxidative modifications of LDL and coronary artery disease (CAD) is suspected but not established. Therefore, the association between plasma levels of oxidized LDL and malondialdehyde (MDA)-modified LDL and acute coronary syndromes and stable CAD was investigated. METHODS and
The study population contained 63 patients with acute coronary syndromes (45 with acute myocardial infarction and 18 with unstable angina pectoris), 35 nontransplanted patients with angiographically confirmed stable angina, 28 heart transplant patients with posttransplant CAD, 79 heart transplant patients without CAD, and 65 control subjects. After correction for age, sex, and LDL and HDL cholesterol, plasma levels of oxidized LDL and MDA-modified LDL were significantly higher in patients with CAD than in individuals without CAD (r2=0.57 and r2=0.26, respectively; both P=0.0001). Plasma levels of MDA-modified LDL were significantly higher in patients with acute coronary syndromes than in individuals with stable CAD (r2=0.65; P=0.0001) and were associated with increased levels of troponin I and C-reactive protein (r2=0.39 and r2=0.34, respectively; both P=0.0001). Plasma levels of oxidized LDL were not associated with increased levels of troponin I and C-reactive protein (r2=0.089 and r2=0.063, respectively).
Elevated plasma levels of oxidized LDL are associated with CAD. Elevated plasma levels of MDA-modified LDL suggest plaque instability and may be useful for the identification of patients with acute coronary syndromes.
Vascular calcification is common in people with diabetes and its presence predicts premature mortality. To clarify the underlying mechanisms, we used low density lipoprotein receptor-deficient (LDLR −/−) mice to study vascular calcification in the ascending aorta. LDLR −/− mice on a chow diet did not develop obesity, diabetes, atheroma, or vascular calcification. In contrast, LDLR −/− mice on high fat diets containing cholesterol developed obesity, severe hyperlipidemia, hyperinsulinemic diabetes, and aortic atheroma. A high fat diet without cholesterol also induced obesity and diabetes, but caused only moderate hyperlipidemia and did not result in significant aortic atheroma formation. Regardless of cholesterol content, high fat diets induced mineralization of the proximal aorta (assessed by von Kossa staining) and promoted aortic expression of Msx2 and Msx1, genes encoding homeodomain transcription factors that regulate mineralization and osseous differentiation programs in the developing skull. Osteopontin(Opn), an osteoblast matrix protein gene also expressed by activated macrophages, was up-regulated in the aorta by these high fat diets. In situ hybridization showed that peri-aortic adventitial cells in high fat-fed mice expressMsx2. Opn was also detected in this adventitial cell population, but in addition was expressed by aortic vascular smooth muscle cells and macrophages of the intimal atheroma.
High fat diets associated with hyperinsulinemic diabetes activate an aortic osteoblast transcriptional regulatory program that is independent of intimal atheroma formation. The spatial pattern of Msx2 and Opn gene expression strongly suggests that vascular calcification, thought to be limited to the media, is an active process that can originate from an osteoprogenitor cell population in the adventitia.
Significant advances in the management of cardiovascular disease have been made possible by the development of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors--"statins." Initial studies explored the impact of statin therapy on coronary artery disease (CAD) progression and regression. Although the angiographic changes were small, associated clinical responses appeared significant. Subsequent large prospective placebo-controlled clinical trials with statins demonstrated benefit in the secondary and primary prevention of CAD in subjects with elevated cholesterol levels. More recently, the efficacy of statins has been extended to the primary prevention of CAD in subjects with average cholesterol levels. Recent studies also suggest that statins have benefits beyond the coronary vascular bed and are capable of reducing ischemic stroke risk by approximately one-third in patients with evidence of vascular disease. In addition to lowering low-density lipoprotein (LDL) cholesterol, statin therapy appears to exhibit pleiotropic effects on many components of atherosclerosis including plaque thrombogenicity, cellular migration, endothelial function and thrombotic tendency. Growing clinical and experimental evidence indicates that the beneficial actions of statins occur rapidly and yield potentially clinically important anti-ischemic effects as early as one month after commencement of therapy. Future investigations are warranted to determine threshold LDL values in primary prevention studies, and to elucidate effects of statins other than LDL lowering. Finally, given the rapid and protean effects of statins on determinants of platelet reactivity, coagulation, and endothelial function, further research may establish a role for statin therapy in acute coronary syndromes.
There is a clear relationship between absolute calcium scores (CS) and severity of coronary artery disease. However, hard coronary events have been shown to occur across all ranges of CS.
We conducted 2 analyses: in group A, 172 patients underwent electron-beam CT (EBCT) imaging within 60 days of suffering an unheralded myocardial infarction. In group B, 632 patients screened by EBCT were followed up for a mean of 32+/-7 months for the development of acute myocardial infarction or cardiac death. The mean patient age and prevalence of coronary calcification were similar in the 2 groups (53+/-8 versus 52+/-9 years and 96% each). In group B, the annualized event rate was 0.11% for subjects with CS of 0, 2.1% for CS 1 to 99, 4.1% for CS 100 to 400, and 4.8% for CS >400, and only 7% of the patients had CS >400. However, mild, moderate, and extensive absolute CSs were distributed similarly between patients with events in both groups (34%, 35%, and 27%, respectively, in group A and 44%, 30%, and 22% in group B). In contrast, the majority of events in both groups occurred in patients with CS >75th percentile (70% in each group).
Coronary calcium is present in most patients who suffer acute coronary events. Although the event rate is greater for patients with high absolute CSs, few patients have this degree of calcification on a screening EBCT. Conversely, the majority of events occur in individuals with high CS percentiles. Hence, CS percentiles constitute a more effective screening method to stratify individuals at risk.
Calcification is almost invariably associated with atherosclerotic plaque lesions. Recent data suggest that plaque calcification is an active, regulated process similar to osteogenesis. In order to clarify the mechanism of plaque calcification, we developed an in vitro model of vascular calcification by utilizing bovine vascular smooth muscle cells (BVSMCs). This model is useful in that diffuse and massive calcification can be induced within 2 weeks and thereby biochemical analyses of vascular calcification can be performed. We have analyzed several aspects of vascular calcification by using this model and demonstrated as follows: 1) in vitro calcification of BVSMCs is regulated by calciotropic hormones and BVSMCs are equipped with a unique autocrine and/or paracrine system regulating calcium metabolism. 2) Sodium-dependent phosphate cotransport plays a crucial role in BVSMC calcification as well as in mineralization of skeletal tissues. 3) BVSMCs acquire osteoblastic phenotype under certain conditions. Finally, we discuss the roles of macrophages in the development of atherosclerotic calcification. Interferon-gamma (IFN-gamma) induces gene expression of 25-hydrovitamin D-1 alpha-hydroxylase (1 alpha OHase) and its activity in macrophages. Since 1 alpha OHase can locally convert 25-hydroxyvitamin D into 1 alpha, 25-dihydroxyvitamin D (1,25(OH)2D), an active metabolite of vitamin D, it is suggested that local production of 1,25(OH)2D by macrophages may promote atherosclerotic calcification. Moreover, macrophages may be involved in the phenotypic changes of vascular smooth muscle cells (VSMCs) to acquire calcifying capacity. Therefore, the phenotypic changes of VSMCs in atherosclerotic plaque may contribute to the development of atherosclerotic calcification.
These studies aim to investigate the expression and function of mineralisation-regulating proteins in association with human vascular calcification focussing on the similarities and differences between the two major calcification pathologies in man: atherosclerotic, intimal calcification and Monckeberg's sclerotic medial calcification.
A number of studies have documented expression of mineralisation-regulating proteins in association with human atherosclerotic calcification leading to the suggestion that human vascular calcification may be a regulated process with similarities to developmental osteogenesis.
In situ hybridisation, immunohistochemistry and semi-quantitative RT-PCR analysis were used to determine the temporal and spatial expression patterns of mineralisation-regulating proteins within human calcified vascular lesions. Additionally, the expression and regulation of bone-associated proteins was analysed during spontaneous calcification of human VSMCs in vitro.
In association with both medial and intimal calcification, the temporal changes in expression of mineralisation-regulating proteins are similar. Some constitutively expressed bone-associated proteins, including matrix Gla protein (MGP), are down-regulated in association with calcification while expression of a number of bone-associated proteins, not normally expressed in the vessel wall, are induced including alkaline phosphatase (ALK), bone sialoprotein (BSP) and bone Gla protein (BGP). In medial calcification the source of expression of these mineralisation-regulating proteins is VSMCs while in intimal lesions both VSMCs and macrophages express them. Furthermore, these bone-associated proteins are spontaneously expressed by VSMCs in vitro suggesting that human VSMCs are capable of simultaneously exhibiting smooth muscle and osteogenic-like properties.
These studies imply that both medial and intimal vascular calcification are regulated processes; however the aetiology of each pathology differs.
The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1-7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.
Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-alpha (TNF-alpha), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-alpha in in vitro vascular calcification.
A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-alpha, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-alpha for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-alpha for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A-specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-alpha. Consistent with this, the intracellular cAMP level was elevated after TNF-alpha treatment. Electrophoretic mobility shift assay demonstrated that TNF-alpha enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation.
These results suggest that TNF-alpha enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.
The aim of this study is to determine whether subpopulations of smooth muscle cells (SMC), as distinguished by variations in contractile and cytoskeletal proteins, appear in the neointima at different times after vascular injury, and/or whether subpopulations develop during serial passaging of these cells. Rat aortae and rabbit carotid arteries were injured with a 2F Fogarty balloon catheter and cultures established from the resulting neointima and the media 2, 6, 12, 16 and 24 weeks later. Cultures were examined at passages 1-5 and subpopulations of SMC categorised by intensity of staining for each protein by immunohistochemistry. Two populations of SMC with different staining intensities ('++', '+') were observed for each of the following proteins: alpha-SM actin, SM-myosin, desmin and vimentin. Populations without these proteins were also found. Changes in the percentages of cells expressing these proteins were transitory, indicating that the populations were not limited to a particular tissue (neointima or media), time after injury or passage number. One exception was found in rabbit cultures where the number of desmin-expressing cells quickly decreased with both time after injury and time in culture. Subpopulations of SMC were found at all times after injury in the media and neointima of rat and rabbit arteries, and after multiple passage of these cells. There was no pattern of development of one population suggesting that either no subpopulation has a proliferative or migratory advantage over others, or that only one population exists that is capable of diverse phenotypic changes.
Increased biomechanical stresses in the fibrous cap of atherosclerotic plaques contribute to plaque rupture and, consequently, to thrombosis and myocardial infarction. Thin fibrous caps and large lipid pools are important determinants of increased plaque stresses. Although coronary calcification is associated with worse cardiovascular prognosis, the relationship between atheroma calcification and stresses is incompletely described.
To test the hypothesis that calcification impacts biomechanical stresses in human atherosclerotic lesions, we studied 20 human coronary lesions with techniques that have previously been shown to predict plaque rupture locations accurately. Ten ruptured and 10 stable lesions derived from post mortem coronary arteries were studied using large-strain finite element analysis. Maximum stress was not correlated with percentage of calcification, but it was positively correlated with the percentage of lipid (P:=0.024). When calcification was eliminated and replaced with fibrous plaque, stress changed insignificantly; the median increase in stress for all specimens was 0.1% (range, 0% to 8%; P:=0.85). In contrast, stress decreased by a median of 26% (range, 1% to 78%; P:=0.02) when lipid was replaced with fibrous plaque.
Calcification does not increase fibrous cap stress in typical ruptured or stable human coronary atherosclerotic lesions. In contrast to lipid pools, which dramatically increase stresses, calcification does not seem to decrease the mechanical stability of the coronary atheroma.
Prediction of hard cardiac events (myocardial infarction and coronary death) remains difficult in spite of the identification of several relevant risk factors for the development of coronary artery disease (CAD). New indicators of risk might add to our predictive ability. We used measures of coronary artery calcification (CAC) found by electron beam tomography (EBT) imaging to develop prediction models for hard cardiac events alone and in association with traditional risk factors for CAD.
Two groups of patients were studied: group A, 676 asymptomatic patients (mean age 52 years, 51% men) prospectively followed up for 32 +/- 7 months after being referred by primary care physicians for a screening EBT, and group B, 10,122 asymptomatic patients screened by EBT at one center and used as controls for calculation of calcium score nomograms.
The occurrence of hard events in group A patients was related to traditional risk factors for CAD, presence of CAC (score >0), Ln (1 + absolute calcium score [CS]), and age- and sex-specific CS percentiles (CS%). Univariate analyses showed that age, smoking, diabetes mellitus, presence of CAC, Ln (1 + absolute CS), and CS% were predictive of hard events (all P <.05). Multiple logistic regression analyses demonstrated that CS% was the only significant predictor of events and provided incremental prognostic value when added to traditional risk factors for CAD (chi-square, P <.001). In a comparison of receiver-operator characteristic curves for prediction of hard events, the area under the curve for CS% plus conventional risk factors and age was significantly larger than that obtained by use of traditional risk factors and age separately as predictors (0.84 vs 0.71, respectively, P <.001). Furthermore, the area under the curve of CS% alone was significantly larger than that of traditional risk factors and age combined (0.82 vs 0.71, P =.028).
Patients are usually selected for EBT screening on the basis of the presence of conventional risk factors for CAD. However, an age- and sex-specific calcium score provides the best predictive model for the occurrence of hard coronary events and adds incremental prognostic information to conventional risk factors for CAD.
For nearly a century, the mechanical failure of calcified heart valves was attributed to a passive degenerative process. Recently, several case reports described bone formation in surgically excised heart valves and suggested an unexpected process of tissue repair.
We studied the prevalence and pathology of heterotopic ossification in 347 surgically excised heart valves (256 aortic, 91 mitral) in 324 consecutive patients (182 men, 142 women; mean age 68 years) who underwent cardiac valve replacement surgery between 1994 and 1998. The valves were examined microscopically to determine the prevalence and features of bone formation and remodeling. Two hundred eighty-eight valves (83%) had dystrophic calcification. Mature lamellar bone with hematopoietic elements and active bone remodeling were present in 36 valves (13%) with dystrophic calcification. Endochondral bone formation, similar to that seen in normal fracture repair, was identified in 4 valves. Microfractures were present in 92% of all valves with ossification. Neoangiogenesis was found in all valves with ossification. Bone morphogenetic proteins 2 and 4 (BMP 2/4), potent osteogenic morphogens, were expressed by myofibroblasts and preosteoblasts in areas adjacent to B- and T-lymphocyte infiltration in valves where ossification was identified. Mast cells were present in calcified and ossified valves and were especially prominent in atheromatous regions.
Heterotopic ossification consisting of mature lamellar bone formation and active bone remodeling is a relatively common and unexpected finding in end-stage valvular heart disease and may be associated with repair of pathological microfractures in calcified cardiac valves.
The purpose of the study was to assess in a prospective design whether plaque morphology is associated with risk of ischemic stroke and other cerebrovascular events in subjects with carotid stenosis.
A total of 223 subjects with carotid stenosis (123 with 35% to 49% degree of stenosis, 100 with 50% to 99% stenosis) and 215 control subjects matched by age and sex who participated in a population health survey at baseline were followed up for 3 years. Plaque echogenicity was assessed by ultrasound at baseline and scored as echolucent, predominantly echolucent, predominantly echogenic, or echogenic. Forty-four subjects experienced >/=1 ischemic cerebrovascular events in the follow-up period. Plaque echogenicity, degree of stenosis, and white blood cell count were independent predictors of cerebrovascular events. The unadjusted relative risk for cerebrovascular events was 13.0 (95% CI 4.5 to 37.4) in subjects with echolucent plaques and 3.7 (95% CI 0.7 to 18.2) in subjects with echogenic plaques when subjects without stenosis were used as the reference. The adjusted relative risk for cerebrovascular events in subjects with echolucent plaques was 4.6 (95% CI 1.1 to 18.9), and there was a significant linear trend (P=0.015) for higher risk with increasing plaque echolucency. The adjusted relative risk for a 10% increase in the degree of stenosis was 1.2 (95% CI 1.04 to 1.4).
Subjects with echolucent atherosclerotic plaques have increased risk of ischemic cerebrovascular events independent of degree of stenosis and cardiovascular risk factors. Subjects at high risk for ischemic vascular events may be identified by ultrasound assessment of plaque morphology.
Over a century ago it was recognized that the vessel wall is a predominant site for ectopic calcification which is a hallmark of clinically significant atherosclerotic lesions. Old observational studies, which characterized vascular calcification as osteogenesis, and recent identification of common molecular mechanisms in bone and vascular calcification have led to the new recognition that atherosclerotic calcification is an actively regulated process similar to osteogenesis and distinct from a metastatic passive mineralization. Since the atherosclerotic lesion is composed of a multitude of cells and inflammatory mediators, elucidation of the role of these components in induction and acceleration of calcification is of fundamental importance in better understanding its pathogenesis and identifying possible interventional targets. This article will focus on four important mediators of vascular calcification: 1) calcifying vascular cells, 2) oxidized lipids, 3) cytokines, and 4) leptin.
Vascular smooth muscle cells (VSMC) exhibit several growth responses to agonists that regulate their function including proliferation (hyperplasia with an increase in cell number), hypertrophy (an increase in cell size without change in DNA content), endoreduplication (an increase in DNA content and usually size), and apoptosis. Both autocrine growth mechanisms (in which the individual cell synthesizes and/or secretes a substance that stimulates that same cell type to undergo a growth response) and paracrine growth mechanisms (in which the individual cells responding to the growth factor synthesize and/or secrete a substance that stimulates neighboring cells of another cell type) are important in VSMC growth. In this review I discuss the autocrine and paracrine growth factors important for VSMC growth in culture and in vessels. Four mechanisms by which individual agonists signal are described: direct effects of agonists on their receptors, transactivation of tyrosine kinase-coupled receptors, generation of reactive oxygen species, and induction/secretion of other growth and survival factors. Additional growth effects mediated by changes in cell matrix are discussed. The temporal and spatial coordination of these events are shown to modulate the environment in which other growth factors initiate cell cycle events. Finally, the heterogeneous nature of VSMC developmental origin provides another level of complexity in VSMC growth mechanisms.
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.
High-resolution magnetic resonance imaging (MRI) with flow suppression not only provides useful information on luminal and wall areas of the carotid artery but also can identify the principal tissue components of the carotid atherosclerotic plaque. The effects of intensive lipid-lowering therapy on these MRI tissue characteristics were examined in patients with coronary disease (CAD). Eight CAD patients who have been receiving intensive lipid-lowering treatment (niacin 2.5 g/d, lovastatin 40 mg/d, and colestipol 20 g/d) for 10 years in the Familial Atherosclerosis Treatment Study (FATS) follow-up were randomly selected from among 60 such treated patients. Eight CAD patients who were matched to the treated patients for age (+/-3 years), baseline low density lipoprotein (+/-5 mg/dL), and triglycerides (+/-50 mg/dL) but who had never been treated with lipid-lowering drugs were selected as controls. For each of these 32 carotid arteries, luminal and plaque areas were measured by planimetry, in a blinded protocol, from the magnetic resonance image that showed most plaque. Fibrous tissue, calcium, and lipid deposits were identified on the basis of established criteria. Plaque composition was estimated as a fraction of total planimetered area. Patients treated with 10-year intensive lipid-lowering therapy, compared with control subjects, had significantly lower low density lipoprotein cholesterol levels (84 versus 158 mg/dL, respectively; P<0.001) and higher high density lipoprotein cholesterol levels (51 versus 37 mg/dL, respectively; P<0.001). As a group, treated patients, compared with untreated control subjects, had a smaller core lipid area (0.7 versus 10.2 mm(2), respectively; P=0.01) and lipid composition (1% versus 17%, respectively). Group differences in luminal area (55 [treated] versus 44 [control] mm(2), P=NS) and plaque area (58 [treated] versus 64 [control] mm(2), P=NS) tended to favor treatment. MRI appears useful for estimating carotid plaque size and composition. Hyperlipidemic CAD patients frequently (97%) have at least moderate (>/=40% area stenosis) carotid plaque. In this case-control study, prolonged intensive lipid-lowering therapy is associated with a markedly decreased lipid content, a characteristic of clinically stable plaques.
In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification.
Calcification is a common complication of atherosclerosis and other chronic inflammatory processes that involves infiltration of monocytes and accumulation of macrophages.
To determine whether these cells modulate vascular calcification in vitro, calcifying vascular cells (CVCs), a subpopulation of osteoblast-like cells derived from the artery wall, were cocultured with human peripheral blood monocytes for 5 days. Results showed that alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation, was significantly greater in cocultures than in cultures of CVCs or monocytes alone. Both ALP activity and matrix mineralization increased in proportion to the number of monocytes added. Activation of monocyte/macrophages (M/Ms) by oxidized LDL further increased ALP activity in cocultures. However, neither conditioned medium from oxidized-LDL-activated M/Ms or transwell coculture had this effect on CVCs, which suggests a need for cell-to-cell contact. In contrast, conditioned medium from lipopolysaccharide-activated M/Ms increased ALP activity of CVCs. ELISA showed that lipopolysaccharide-activated M/Ms secreted tumor necrosis factor-alpha, and neutralizing antibody to tumor necrosis factor-alpha attenuated the induction of ALP activity by the conditioned media.
These results suggest that M/Ms enhance in vitro vascular calcification via 2 independent mechanisms: cell-cell interaction and production of soluble factors such as tumor necrosis factor-alpha.
Bone formation and dystrophic calcification are present in carotid endarterectomy plaques. The clinical significance of these findings is unknown. The purpose of this study was to determine whether bone formation and extensive dystrophic calcification are associated with stable plaques and protective against ischemic vascular events.
Carotid endarterectomy plaques were collected from 142 patients (94 men) with carotid stenosis. The specimens were evaluated for lamellar bone formation, dystrophic calcifications, inflammatory infiltrates, neovascularization, and histological type or grade of plaque according to a standard AHA grading system. Immunohistochemical staining was performed to identify vascular endothelial cells in neovascularization (factor VIII) and lymphocytes. Clinical data, including history of cerebrovascular and cardiovascular events, were recorded at the time of surgery.
Patients with calcification of carotid plaques had fewer symptoms of stroke and transient ischemic attack (P=0.042) than those without calcification. Stroke and transient ischemic attack occurred less frequently in patients with plaques with large calcific granules (P=0.021). Of the patients, 13% had lamellar bone formation, which directly correlated with the presence of sheetlike calcifications (P=0.0001) and inversely correlated with ulcerated lesions (P=0.048). The presence of bone also correlated with diabetes (P<0.01) and coronary artery disease (P<0.01). Of the 20 patients with bone, 6 had a history of stoke and transient ischemic attack (P=0.5).
The results indicate that bone formation tends to occur in heavily calcified carotid lesions devoid of ulceration and hemorrhage. Patients with extensive calcification of the carotid plaques are less likely to have symptomatic disease.
Phosphate regulation of vas-cular smooth muscle cell calcification
- S Jono
- Md Mckee
- Murry
- Ce
Jono S, McKee MD, Murry CE, et al. Phosphate regulation of vas-cular smooth muscle cell calcification Circ Res 2000;87:E10 –7.
Ath-erosclerosis and coronary artery disease. Philadelphia: Lippincott-Raven
- Hc Mcgill
- Overview
- Ross V R Infuster
- Topol
McGill HC. Overview. InFuster V, Ross R, Topol EJ, editors. Ath-erosclerosis and coronary artery disease. Philadelphia: Lippincott-Raven; 1996. p. 25– 41.