Involvement of matrix metalloproteinase-2 in the development of medial layer vascular calcification in uremic rats

Vascular calcification is the most important cause of cardiovascular disease in patients with chronic kidney disease (CKD). Medial layer vascular calcification, which is recognized to be an active process (i.e. the transformation of vascular smooth muscle cells into osteoblast-like cells), is common in CKD patients. We have recently reported the possibility of an interaction between elastin degradation and medial layer vascular calcification. Matrix metalloproteinase-2 (MMP-2), which induces the degradation of elastin, has been implicated in the elastic calcification in arteries of dialysis patients; however, the precise mechanisms by which elastin degradation interacts with the development of vascular calcification remain to be studied. To clarify the mechanisms by which elastin degradation is involved in the development of medial layer vascular calcification in the uremic milieu, we induced aortic medial layer calcification in 5/6 nephrectomized uremic rats (male Sprague-Dawley rats) fed a diet containing high phosphate (1.2%) and lactate (20%). After 10 weeks, the rats were euthanized for the measurement of serum chemistry profiles and histological analyses. The uremic rats showed significant increases in blood pressure, serum creatinine, phosphate, and parathyroid hormone levels compared with normal rats. Von Kossa staining showed medial layer aortic calcification in the uremic rats. In calcified lesions, thin elastic lamellae were observed by elastin staining, indicating that elastin degradation could occur in the area. Furthermore, MMP-2 expression determined by immunohistochemistry was also observed in the same area. Elastin degradation accompanied by MMP-2 expression might be involved in the development of medial layer vascular calcification in uremic rats.     

Elastosis in diverticular disease of the sigmoid colon.

Diverticular disease of the sigmoid colon is an increasingly common clinical problem in the ageing population of western industrialised countries but the mechanism by which the disease develops remains unknown. The muscular abnormality is the most striking and consistent feature and this has been studied by light and electron microscopy in 25 surgical specimens of uncomplicated diverticular disease and in 25 controls. This is the first ultrastructural study of human colonic muscle to be published and shows that the muscle cells in diverticular disease are normal; neither hypertrophy nor hyperplasia is present. There is, however, an increase in the elastin content of the taeniae coli by greater than 200% compared with controls: elastin is laid down between the muscle cells and the normal fascicular pattern of the taeniae coli is distorted. There is no alteration in the elastin content of the circular muscle. As elastin is laid down in a contracted form, this elastosis may be responsible for the shortening or 'contracture' of the taeniae which in turn leads to the characteristic concertina-like corrugation of the circular muscle. Such a structural change could explain the altered behaviour of the colon wall in diverticular disease and its failure to change on treatment with bran.     

Elastin biosynthesis: The missing link in tissue-engineered blood vessels

Nearly 20 years have passed since Weinberg and Bell attempted to make the first tissue-engineered blood vessels. Following this early attempt, vascular tissue engineering has emerged as one of the most promising approaches to fabricate orderly and mechanically competent vascular substitutes. In elastic and muscular arteries, elastin is a critical structural and regulatory matrix protein and plays an important and dominant role by conferring elasticity to the vessel wall. Elastin also regulates vascular smooth muscle cells activity and phenotype. Despite the great promise that tissue-engineered blood vessels have to offer, little research in the last two decades has addressed the importance of elastin incorporation into these vessels. Although cardiovascular tissue engineering has been reviewed in the past, very little attention has been given to elastin. Thus, this review focuses on the recent advances made towards elastogenesis and the challenges we face in the quest for appropriate functional vascular substitutes.     

Elastin haploinsufficiency induces alternative aging processes in the aorta

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.     

Effect of an elastin growth substrate on cholesteryl ester synthesis and foam cell formation by cultured aortic smooth muscle cells

Exposure of smooth muscle cells cultured on plastic or glass to hyperlipidemic serum did not result in the formation of foam cells. Since elastin binds serum lipids, and vascular smooth muscle cells are normally closely associated with elastin, we investigated the effects of an elastin substrate on lipid metabolism and on the accumulation of lipid vacuoles by rabbit aortic smooth muscle cells in culture. When cells were grown in plastic petri dishes, cholesteryl ester synthesis, as measured by [14C]oleate incorporation into cholesteryl esters, was 3 times greater in rabbit hyperlipidemic serum (HLS) than in normolipemic serum (NLS) (P less than 0.001). For cells of the same subculture grown on the elastin substrate, the synthetic rate was 6-fold greater in HLS compared to NLS (P less than 0.005). The cells grown on the elastin membranes in the presence of HLS contained large numbers of Oil red O stainable lipid vacuoles and resembled foam cells, while those grown in petri dishes and exposed to HLS showed only an occasional cell containing a few vacuoles. Pre-incubation in lipoprotein-deficient serum markedly enhanced the stimulatory effect of HLS on cholesteryl ester synthesis for cells growing in plastic petric dishes but had much less stimulatory effect on the cells growing on elastin membranes. These studies indicate that close association with elastin modulates the response of smooth muscle cells to hyperlipidemia and suggest a role for elastin in the formation of foam cells of smooth muscle origin during atherogenesis.     

Vascular remodeling and media calcification increases arterial stiffness in chronic kidney disease

Background: Cardiovascular disease is the most common cause of death in patients with chronic kidney disease (CKD). Arterial stiffness and calcification are non-traditional risk factors of cardiovascular disease in CKD. In CKD rats, we investigated the involvement of smooth muscle cells differentiation to osteoblast-like cells and blood vessel wall remodeling, associated with media calcification, in arterial stiffness.

Method: CKD with vascular calcification was induced by subtotal nephrectomy followed by treatment with a high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). At week 3–6, hemodynamic parameters and pulse wave velocity (PWV) were assessed. Vascular media calcification and remodeling were determined by histological von Kossa staining and confocal immunofluorescence analysis of osteocalcin, elastin, α-smooth muscle actin (α-SMA) and collagen-1.

Results: Treatment of CKD rats with Ca/P/VitD, but not normal animals, induced a significant increase in pulse pressure and PWV (p?de novo expression of osteocalcin was observed, whereas α-SMA immunofluorescence levels were reduced (p?p?Conclusion: This study indicate that smooth muscle cells differentiation to osteoblast-like cells and the associated media remodeling, which includes disruption of elastic lamellas and deposition of collagen are, at least in part, associated with the increased arterial stiffness observed in CKD rats with vascular calcification.     

Elastin fragmentation and atherosclerosis progression: The elastokine concept

Atherosclerosis is a progressive multifaceted inflammatory disease affecting large- and medium-sized arteries. Typical feature of this disease is the formation and build-up of atherosclerotic plaques characterized by vascular extracellular matrix degradation and remodeling. Many studies have documented degradation of native elastin, the main extracellular matrix protein responsible for resilience and elasticity of arteries, by local release of elastases, leading to the production of elastin-derived peptides (EDP). These peptides have been proposed to actively participate in the progression of the disease by accelerating different biological processes, such as LDL oxidation and calcification of the vascular wall. These pathophysiological effects are mediated by the binding of EDP on a peculiar heterotrimeric receptor named elastin receptor complex (ERC). In this article, we review the contribution of elastin in biological processes involved in atherosclerosis progression from its initial elastase-driven degradation to its ultimate cellular effects. Finally, we discuss the ERC and its derived signaling pathways as promising therapeutic targets.     

Elastin and vascular disease

Supravalvular aortic stenosis (SVAS) is a vascular disease that primarily affects large arteries, like the aorta and pulmonary arteries. SVAS can be inherited as an isolated, autosomal dominant trait or as part of a complex developmental disorder, Williams syndrome. Molecular genetic studies indicate that mutations affecting part of an elastin allele cause autosomal dominant SVAS while submicroscopic deletions that disrupt the entire elastin gene (and presumably adjacent loci) are responsible for Williams syndrome. These studies suggest that loss of vascular elasticity from any cause may contribute to vascular obstruction.     

Small Artery Elastin Distribution and Architecture—Focus on Three Dimensional Organization

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet‐like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub‐serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three‐dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions.     

Elastin in asthma

Extracellular matrix is generally increased in asthma, causing thickening of the airways which may either increase or decrease airway responsiveness, depending on the mechanical requirements of the deposited matrix. However, in vitro studies have shown that the altered extracellular matrix produced by asthmatic airway smooth muscle cells is able to induce increased proliferation of non-asthmatic smooth muscle cells, which is a process believed to contribute to airway hyper-responsiveness in asthma. Elastin is an extracellular matrix protein that is altered in asthmatic airways, but there has been no systematic investigation of the functional effect of these changes. This review reveals divergent reports of the state of elastin in the airway wall in asthma. In some layers of the airway it has been described as increased, decreased and/or fragmented, or unchanged. There is also considerable evidence for an imbalance of matrix metalloproteinases, which degrade elastin, and their respective inhibitors the tissue inhibitors of metalloproteinases, which collectively help to explain observations of both increased elastin and elastin fragments. A loss of lung elastic recoil in asthma suggests a mechanical role for disordered elastin in the aetiology of the disease, but extensive studies of elastin in other tissues show that elastin fragments elicit cellular effects such as increased proliferation and inflammation. This review summarises the current understanding of the role of elastin in the asthmatic airway.     

Elastin degradation is associated with progressive aortic stiffening and all-cause mortality in predialysis chronic kidney disease

In the large conduit arteries, elastin is important in maintaining vascular compliance. Studies in animal models suggest that elastin degradation may promote arteriosclerotic vascular changes. There is already a well-established link between aortic stiffening and mortality in the general population and in patients undergoing dialysis. Elastin degradation is mediated by several proteases, including matrix metalloproteinase 2 and cathepsin S. Elastin turnover may be inferred by measuring serum levels of elastin-derived peptides. We analyzed the serum concentration of these biomarkers, their endogenous inhibitors, and aortic pulse wave velocity in 200 patients with stages 3 and 4 chronic kidney disease and then serially in a subgroup of 65 patients over 36 months. Serum matrix metalloproteinase 2, cathepsin S, and elastin-derived peptide levels were independently associated with baseline aortic pulse wave velocity and changes in stiffness over the follow-up period. Higher matrix metalloproteinase 2 and elastin-derived peptide levels were also independently associated with preexisting cardiovascular disease. In multivariable Cox regression, higher serum elastin-derived peptide levels were independently associated with increased all-cause mortality (hazard ratio per SD increase=1.78; P=0.021). In predialysis chronic kidney disease, elastin degradation is an important determinant of arterial stiffness and is associated with all-cause mortality.     

The mechanical performance and histomorphological structure of the descending aorta in hyperthyroidism

Thyroid hormones decrease systemic vascular resistance by directly affecting vascular smooth muscle relaxation. There is limited literature about their effect on the mechanical performance of the aortic wall. Therefore, the authors determined the influence of hyperthyroidism on the mechanical properties and histomorphological structure of the descending thoracic aorta in rats. Severe hyperthyroidism was induced in 20 male Wistar rats by administering L-thyroxine (T(4)) in their drinking water for 8 weeks; age-matched normal euthyroid rats acted as controls. Animals were sacrificed, and the mechanical and histomorphometrical characteristics of the descending thoracic aorta were studied. The aortic wall of hyperthyroid rats was stiffer than that of euthyroid animals at the upper physiologic levels of stress or strain (p < 0.05) but less stiff at the lower physiologic and lower levels (p < 0.05). The aorta of hyperthyroid animals compared with that of euthyroid ones showed an increase of the internal and external diameters (p < 0.05), the media area (p < 0.05), the number of smooth muscle cell nuclei (p < 0.05), and the collagen density (p < 0.05) and a decrease in the elastin laminae thickness (p < 0.001) and elastin density (p < 0.001). In hyperthyroid rats, the aortic wall was stiffer at the upper physiologic and higher levels of stress and strain. These changes correlated with microstructural changes of the aortic wall. The coexistence of hyperthyroidism with disease states or clinical conditions that predispose to increased arterial pressure may be associated with increased arterial stiffness and have undesirable consequences on the mechanical performance of the thoracic aorta and hemodynamic homeostasis. These changes could lead to an increased risk for developing vascular complications.     

Lysosomal cysteine proteases in atherosclerosis

Atherosclerosis is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathologic events, recent data from atherosclerotic patients and animals suggest the participation of lysosomal cysteine proteases in atherogenesis. Atherosclerotic lesions in humans overexpress the elastolytic and collagenolytic cathepsins S, K, and L but show relatively reduced expression of cystatin C, their endogenous inhibitor, suggesting a shift in the balance between cysteine proteases and their inhibitor that favors remodeling of the vascular wall. Extracts of human atheromatous tissue show greater elastolytic activity in vitro than do those from healthy donors. The cysteinyl protease inhibitor E64d limits this increased elastolysis, indicating involvement of cysteine proteases in elastin degradation during atherogenesis. Furthermore, inflammatory cytokines augment expression and secretion of active cysteine proteases from cultured monocyte-derived macrophages, vascular smooth muscle cells, and endothelial cells and increase degradation of extracellular elastin and collagen. Cathepsin S-deficient cells or those treated with E64d show significantly impaired elastolytic or collagenolytic activity. Additionally, recent in vivo studies of atherosclerosis-prone, LDL receptor-null mice lacking cathepsin S show participation of this enzyme in the initial infiltration of leukocytes, medial elastic lamina degradation, endothelial cell invasion, and neovascularization, illustrating an important role for cysteine proteases in arterial remodeling and atherogenesis.     

Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration

Fibulin (fbln)-5 is an elastin-binding protein required for assembly and organization of elastic fibers. To examine the potential role of fbln-5 in vascular remodeling and neointima formation, we induced vascular injury by carotid artery ligation in fbln-5(-/-) mice. Mutant mice displayed an exaggerated vascular remodeling response that was accompanied by severe neointima formation with thickened adventitia. These abnormalities were not observed in elastin(+/-) mice that exhibited a comparable reduction of vessel extensibility to fbln-5(-/-) mice. Thus, the severe remodeling response could not be attributed to altered extensibility of the vessel wall alone. Vascular smooth muscle cells cultured from fbln-5(-/-) mice displayed enhanced proliferative and migratory responses to mitogenic stimulation relative to wild-type cells, and these responses were inhibited by overexpression of fbln-5. These findings demonstrate the importance of the elastic laminae in vascular injury, and reveal an unexpected role of fbln-5 as an inhibitor of vascular smooth muscle cell proliferation and migration.     

Atherosclerosis and matrix dystrophy

Atherosclerosis is characterized by inflammatory metabolic change with lipid accumulation in the artery. Atherosclerotic plaque occurs at discrete locations in the arterial system and involves the proliferation of smooth muscle cells (SMCs) together with imbalance of the extracellular matrix elements, elastic fiber in particular. The role of elastin in arterial development and disease was confirmed by generating mice that lack elastin. Thus, elastin is a critical regulatory molecule that regulates the phenotypic modulation, proliferation and migration of SMCs. We estimated that elastin expression and SMC proliferation are coupled inversely: potent stimulators of cell proliferation may potentially inhibit elastin expression and potent inhibitors of cell proliferation can stimulate elastin expression. Moreover, elastin was found to be expressed maximally at the G(0) and minimally at the G(2)/M phase during the cell cycle, suggesting that its expression is regulated by the cell growth state. The elastin peptide VPGVG enhanced SMC proliferation, resulting in the reduction of elastin expression. The inhibition of elastin expression by elastin fragments may be reflected in the negative feedback regulatory mechanism. The relationship between cell proliferation and elastin expression may be changed in atherosclerosis. Areas of atherosclerotic plaque show abnormality of elasticity and permeability from the viewpoint of the physiological function of the arterial wall. The etiology was estimated to be that cholesterol and calcium are deposited on the elastic fiber, resulting in decreased elastin synthesis and cross-linking formation. In addition, these dysfunctions of elastin fiber are also associated, in that the down-regulation of elastin and its related components (fibrillin-1 and lysyl oxidase) are directly related to calcification in SMCs. The denatured arterial elastin by cholesterol and calcium accumulation was also susceptible to proteolytic enzymes such as elastase and matrix metalloproteinase (MMP). Therefore, metabolic change in elastic fiber induces decreased elasticity and is associated with essential hypertension. Vitamin K(2) is used in drug therapy against atherosclerosis, or calcification in diabetes mellitus or dialysis, due to its promotion of the carboxylation of the matrix Gla protein.     

Coronary benefits of calcium antagonist therapy for patients with hypertension.

Calcium antagonists effective in lowering blood pressure are a heterogeneous group including three main classes: phenylalkylamines, benzothiazepines and dihydropyridines. Dihydropyridines have a dual mode of action upon the endothelium contributing to their beneficial antihypertensive effects: (1) direct relaxation by inhibition of smooth muscle L-type calcium current, and (2) indirect relaxation through release of nitric oxide from the vascular endothelium. Calcium antagonists may affect many calcium-dependent events in the formation of atherosclerosis such as the localized accumulation of collagen, elastin, and calcium together with monocyte infiltration and smooth muscle proliferation and migration. In the INSIGHT calcification study, the overall treatment effect of nifedipine demonstrated significant inhibition of coronary calcium progression over a three-year period. Calcium antagonists improve symptoms and reduce ischemia in hypertensive patients with ischemic heart disease. Although in placebo-controlled trials calcium antagonists demonstrated a significant reduction in cardiovascular morbidity and mortality, they may be less effective than other types of antihypertensive drugs in preventing ischemic heart disease.     

Pathology of the ageing--diverticular disease

The correlation of increased incidence of diverticular disease with age is well documented. Such a correlation results from the development of a structural change in the taeniae coli, a progressive elastosis. The consequences of this elastosis are a shortening of the taeniae coli and a subsequent change in the circular muscle layer secondary to this. This type of structural alteration takes time to develop and thus explains the time lag experienced between a change in diet and an altered incidence of the disease. Eastwood et al (1982) have suggested that diverticular disease is merely a normal concomitant of ageing which is degenerative in nature. However, the changes in structure in this condition appear to be dynamic, being associated with an altered intraluminal environment. Such a concept is crucial to our understanding of the pathology of ageing in general. Atherogenesis is associated with muscle cell hypertrophy, another dynamic change, which also leads to elastin formation. This suggests that treatment of such conditions should not just be limited to the control of an inevitable deterioration but should be directed to the investigation of the stimuli that may trigger such conditions. For example, it is interesting to speculate exactly when the changes that lead to diverticular disease begin: not only is a high fibre diet eaten in Africa, but breast-feeding may continue until the age of two years. The greatest increase in thickness in the normal colon occurs in this period and it may be that early weaning distorts this proliferation. The initiating factor in the aetiology of elastogenesis could be the small stools produced on a 'Western' diet which only intermittently distend the colon. Arterial smooth muscle cells increase their uptake of elastin precursors (particularly proline) when subjected to intermittent distension (Leung et al, 1976) and this may form a common link in the changes generated in vascular and colonic muscle tissue with time. This type of change is independent of alterations in motility and thus explains why asymptomatic patients have a normal motility index (Weinreich and Anderson, 1976). The muscular thickening in uncomplicated diverticular disease can therefore be explained in terms of elastosis and contracture of the taeniae coli in the presence of normal muscle cells. This does not exclude the possibility that hypertrophy and hyperplasia of these cells can develop in response to subsequent pericolic inflammation and fibrosis.     

Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome

Marfan syndrome is associated with early death due to aortic aneurysm. The condition is caused by mutations in the gene (FBN1) encoding fibrillin-1, a major constituent of extracellular microfibrils. Prior observations suggested that a deficiency of microfibrils causes failure of elastic fiber assembly during late fetal development. Mice homozygous for a targeted hypomorphic allele (mgR) of Fbn1 revealed a predictable sequence of abnormalities in the vessel wall including elastic fiber calcification, excessive deposition of matrix elements, elastolysis, and intimal hyperplasia. Here we describe previously unrecognized concordant findings in elastic vessels from patients with Marfan syndrome. Furthermore, ultrastructural analysis of mgR mice revealed cellular events that initiate destructive changes. The first detectable abnormality was an unusually smooth surface of elastic laminae, manifesting the loss of cell attachments that are normally mediated by fibrillin-1. Adjacent cells adopted alteration in their expression profile accompanied by morphological changes but retained expression of vascular smooth muscle cell markers. The abnormal synthetic repertoire of these morphologically abnormal smooth muscle cells in early vascular lesions included elastin, among other matrix elements, and matrix metalloproteinase 9, a known mediator of elastolysis. Ultimately, cell processes associated with zones of elastic fiber thinning and fragmentation. These data suggest that the loss of cell attachments signals a nonproductive program to synthesize and remodel an elastic matrix. This refined understanding of the pathogenesis of vascular disease in Marfan syndrome will facilitate the development of therapeutic strategies.     

Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.     

Biology of calcification in vascular cells: intima versus media

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