elastin and Vascular-Calcification

Aortic aneurysm is a vascular disease whereby the ECM (extracellular matrix) of a blood vessel degenerates, leading to dilation and eventually vessel wall rupture. Recently, it was shown that calcification of the vessel wall is involved in both the initiation and progression of aneurysms. Changes in aortic wall structure that lead to aneurysm formation and vascular calcification are actively mediated by vascular smooth muscle cells. Vascular smooth muscle cells in a healthy vessel wall are termed contractile as they maintain vascular tone and remain quiescent. However, in pathological conditions they can dedifferentiate into a synthetic phenotype, whereby they secrete extracellular vesicles, proliferate, and migrate to repair injury. This process is called phenotypic switching and is often the first step in vascular pathology. Additionally, healthy vascular smooth muscle cells synthesize VKDPs (vitamin K-dependent proteins), which are involved in inhibition of vascular calcification. The metabolism of these proteins is known to be disrupted in vascular pathologies. In this review, we summarize the current literature on vascular smooth muscle cell phenotypic switching and vascular calcification in relation to aneurysm. Moreover, we address the role of vitamin K and VKDPs that are involved in vascular calcification and aneurysm. Visual Overview- An online visual overview is available for this article.

Topics: Aortic Aneurysm; Elastin; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oxidative Stress; Phenotype; Transforming Growth Factor beta; Vascular Calcification; Vitamin K; Vitamin K Epoxide Reductases

Vascular calcification is an independent risk factor for the development of cardiovascular disease and is classified into two types based on the site of calcification : intimal atherosclerotic calcification and Mönckeberg's medial calcification. Matrix vesicles released from macrophages and vascular smooth muscle cells (VSMC) during apoptosis play a pivotal role in formation of fine granular calcification, while osteogenic differentiation of VSMC contributes to progression of advanced calcification. Recent noninvasive imaging studies of atherosclerotic calcification provide robust evidence that inflammation precedes active calcification, leading to establish the inflammation-dependent calcification paradigm. On the other hand, elastin degradation by increased elastolytic activities and disturbance of regulatory systems of extracellular pyrophosphate metabolism play an important role in development of Mönckeberg's medial calcification.

Topics: Animals; Apoptosis; Calcinosis; Cardiovascular Diseases; Cellular Senescence; Diphosphates; Elastin; Extracellular Matrix; Humans; Macrophages; Mice; Muscle, Smooth, Vascular; Osteogenesis; Risk Factors; Vascular Calcification

Patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) have significant cardiovascular morbidity and mortality that is in part due to the development of vascular calcification. Vascular calcification is an active, highly regulated process that shares many similarities with normal bone formation. New discoveries related to extracellular vesicles, microRNAs, and calciprotein particles continue to reveal the mechanisms that are involved in the initiation and progression of vascular calcification in CKD. Further innovations in these fields are critical for the development of biomarkers and therapeutic options for patients with CKD and ESRD.

Topics: Calcium; Disease Progression; Elastin; Humans; Kidney Failure, Chronic; Muscle, Smooth, Vascular; Phosphorus; Renal Insufficiency, Chronic; Risk Factors; Vascular Calcification

Extracellular matrix (ECM) proteins have been associated with atherosclerotic complications, such as plaque rupture, calcification and aneurysm. It is not clear what role different types of collagen play in the pathomechanism of atherosclerosis. The aim of the study was to analyze the content of elastin and major types of collagen in the aortic wall and how they associated are with course of atherosclerosis.. In this work we present six biochemical parameters related to ECM proteins and collagen-specific amino acids (collagen type I, III, and IV, elastin, proline and hydroxyproline) analyzed in 106 patients' aortic wall specimens characterized by different degree of atherosclerosis. Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS), ELISA and immunohistochemical methods were used. The severity of atherosclerosis was assessed on the six-point scale of the American Heart Association, taking into account the number and location of foam cells, the presence of a fatty core, calcium deposits and other characteristic atherosclerotic features.. The results show that there is a relationship between the content of collagen-specific amino acids and development of atherosclerosis. The degree of atherosclerotic lesions was negatively correlated with the content of proline, hydroxyproline and the ratio of these two amino acids. Calcium deposits and surrounding tissue were compared and it was demonstrated that the ratio of type I collagen to type III collagen was higher in the aortic tissue than in aortic calcification areas, while the ratio of collagen type III to elastin was smaller in the artery than in the calcium deposits.. We suggest that increase in collagen type III presence in the calcification matrix may stem from disorders in the structure of the type I and III collagen fibers. These anomalous fibers are likely to favor accumulation of the calcium salts, an important feature of the process of atheromatosis.

Topics: Adult; Aged; Aorta; Atherosclerosis; Collagen; Elastin; Female; Humans; Hydroxyproline; Male; Middle Aged; Proline; Vascular Calcification

Peritoneal dialysis (PD) patients experience accelerated arterial aging, which is characterized by elastin degradation. Elastin-derived peptides (EDPs) are direct products of elastin fragmentation. This study tried to explore the association between serum EDPs and abdominal aortic calcification (AAC) in PD patients.. Serum levels of EDPs were analyzed in 126 eligible PD patients and 30 controls. PD patients were grouped according to the annularity of AAC evaluated by an abdominal computed tomography (CT) scan. Serum EDPs were analyzed in relation to the presence of AAC or severe AAC in PD patients by logistic regression analysis.. Serum EDPs in PD patients were significantly higher than age-matched controls. In 126 PD patients, higher EDPs was associated with greater risk of present AAC (OR = 1.056, 95%CI 1.010-1.103) and severe AAC (OR = 1.062, 95%CI 1.004-1.123). A combination of EDPs substantially improved the accuracy of diagnostic performance for AAC and severe AAC.. EDPs can predict the presence and extent of AAC in PD patients, indicating its possible role to recognize PD patients at risk for AAC and severe AAC.

Topics: Adult; Aorta, Abdominal; Case-Control Studies; Cross-Sectional Studies; Elastin; Female; Humans; Logistic Models; Male; Middle Aged; Peptide Fragments; Peritoneal Dialysis; Predictive Value of Tests; Risk Factors; Tomography, X-Ray Computed; Vascular Calcification

Inflammation and stiffness in the arteries is referred to as vascular calcification. This process is a prevalent yet poorly understood consequence of cardiovascular disease and diabetes mellitus, comorbidities with few treatments clinically available. Because this is an active process similar to bone formation, it is hypothesized that osteoclasts (OCs), bone-resorbing cells in the body, could potentially work to reverse existing calcification by resorbing bone material. The receptor activator of nuclear kappa B-ligand (RANKL) is a molecule responsible for triggering a response in monocytes and macrophages that allows them to differentiate into functional OCs. In this study, OC and RANKL delivery were employed to determine whether calcification could be attenuated. OCs were either delivered via direct injection, collagen/alginate microbeads, or collagen gel application, while RANKL was delivered via injection, through either a porcine subdermal model or aortic injury model. While in vitro results yielded a decrease in calcification using OC therapy, in vivo delivery mechanisms did not provide control or regulation to keep cells localized long enough to induce calcification reduction. However, these results do provide context and direction for the future of OC therapy, revealing necessary steps for this treatment to effectively reduce calcification in vivo. The discrepancy between in vivo and in vitro success for OC therapy points to the need for a more stable and time-controlled delivery mechanism that will allow OCs not only to remain at the site of calcification, but also to be regulated so that they are healthy and functioning normally when introduced to diseased tissue.

Topics: Animals; Bone Resorption; Carrier Proteins; Cell Differentiation; Cell- and Tissue-Based Therapy; Cells, Cultured; Elastin; Macrophages; Male; Membrane Glycoproteins; Monocytes; Myocytes, Smooth Muscle; Osteoclasts; Osteogenesis; RANK Ligand; Rats; Rats, Sprague-Dawley; Receptor Activator of Nuclear Factor-kappa B; Swine; Vascular Calcification

Generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE) are characterized by pathologic calcifications in the media of large- and medium sized arteries. GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. Different treatment approaches including bisphosphonates and orally administered pyrophosphate (PP

Topics: Animals; Antibodies; Aorta; Calcium Chelating Agents; Cell Line; Drug Carriers; Drug Compounding; Elastin; Female; Humans; Male; Mice, Inbred C57BL; Mice, Knockout; Multidrug Resistance-Associated Proteins; Nanoparticles; Pentetic Acid; Pseudoxanthoma Elasticum; Serum Albumin, Human; Vascular Calcification

Using maximum diameter of an abdominal aortic aneurysm (AAA) alone for management can lead to delayed interventions or unnecessary urgent repairs. Abdominal aortic aneurysm stiffness plays an important role in its expansion and rupture. In vivo aortic magnetic resonance elastography (MRE) was developed to spatially measure AAA stiffness in previous pilot studies and has not been thoroughly validated and evaluated for its potential clinical value. This study aims to evaluate noninvasive in vivo aortic MRE-derived stiffness in an AAA porcine model and investigate the relationships between MRE-derived AAA stiffness and (1) histopathology, (2) uniaxial tensile test, and (3) burst testing for assessing MRE's potential in evaluating AAA rupture risk.. Abdominal aortic aneurysm was induced in 31 Yorkshire pigs (n = 226 stiffness measurements). Animals were randomly divided into 3 cohorts: 2-week, 4-week, and 4-week-burst. Aortic MRE was sequentially performed. Histopathologic analyses were performed to quantify elastin, collagen, and mineral densities. Uniaxial tensile test and burst testing were conducted to measure peak stress and burst pressure for assessing the ultimate wall strength.. Magnetic resonance elastography-derived AAA stiffness was significantly higher than the normal aorta. Significant reduction in elastin and collagen densities as well as increased mineralization was observed in AAAs. Uniaxial tensile test and burst testing revealed reduced ultimate wall strength. Magnetic resonance elastography-derived aortic stiffness correlated to elastin density (ρ = -0.68; P < 0.0001; n = 60) and mineralization (ρ = 0.59; P < 0.0001; n = 60). Inverse correlations were observed between aortic stiffness and peak stress (ρ = -0.32; P = 0.0495; n = 38) as well as burst pressure (ρ = -0.55; P = 0.0116; n = 20).. Noninvasive in vivo aortic MRE successfully detected aortic wall stiffening, confirming the extracellular matrix remodeling observed in the histopathologic analyses. These mural changes diminished wall strength. Inverse correlation between MRE-derived aortic stiffness and aortic wall strength suggests that MRE-derived stiffness can be a potential biomarker for clinically assessing AAA wall status and rupture potential.

Topics: Animals; Aortic Aneurysm, Abdominal; Collagen; Disease Models, Animal; Elasticity Imaging Techniques; Elastin; Swine; Vascular Calcification; Vascular Stiffness

Calcium phosphate minerals deposit on the elastin-rich medial layers of arteries in the majority of seniors, diabetic, and chronic kidney disease patients, causing severe cardiovascular complications. There is no cure for medial calcification, and the mechanism of mineral formation on elastin layers is unknown. Here we propose cross-linked elastin-like polypeptide membranes as models to study medial calcification. Calcium phosphates deposit first on fibers and filaments and then spread to globular structures present in the membranes. Mineral phase evolution analyzed by near-edge X-ray spectroscopy matches that previously observed in a mouse model of medial calcification, showing that this simple system captures some of the key in vivo findings. This work shows how minerals form and evolve upon nucleation on elastin and provides an in vitro model that can be tuned to study hypotheses related to arterial calcification mechanisms and test drugs to stop or revert mineralization.

Topics: Animals; Elastin; Humans; Membranes, Artificial; Mice; Models, Cardiovascular; Vascular Calcification

Elastin plays an important role in maintaining blood vessel integrity. Proteolytic degradation of elastin in the vascular system promotes the development of atherosclerosis, including blood vessel calcification. Cysteine cathepsins have been implicated in this process, however, their role in disease progression and associated complications remains unclear. Here, we showed that the degradation of vascular elastin by cathepsins (Cat) K, S, and V directly stimulates the mineralization of elastin and that mineralized insoluble elastin fibers were ~25-30% more resistant to CatK, S, and V degradation when compared to native elastin. Energy dispersive X-ray spectroscopy investigations showed that insoluble elastin predigested by CatK, S, or V displayed an elemental percentage in calcium and phosphate up to 8-fold higher when compared to non-digested elastin. Cathepsin-generated elastin peptides increased the calcification of MOVAS-1 cells acting through the ERK1/2 pathway by 34-36%. We made similar observations when cathepsin-generated elastin peptides were added to ex vivo mouse aorta rings. Altogether, our data suggest that CatK-, S-, and V-mediated elastolysis directly accelerates the mineralization of the vascular matrix by the generation of nucleation points in the elastin matrix and indirectly by elastin-derived peptides stimulating the calcification by vascular smooth muscle cells. Both processes inversely protect against further extracellular matrix degradation.

Topics: Animals; Aorta; Cathepsins; Cells, Cultured; Elastin; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proteolysis; Vascular Calcification

The development of calcification-resistant bioprosthetic materials is a very important challenge for paediatric surgery. The subcutaneous implantation in rats is the well-known first-stage model for this kind of research. Using this model, we aimed to compare calcification of the porcine aortic wall and bovine pericardium and jugular vein wall cross-linked with glutaraldehyde (GA) and ethylene glycol diglycidyl ether (DE). We also determined the efficacy of DE-preserved tissue modification with 2-(2-carboxyethylamino)ethylidene-1,1-bisphosphonic acid (CEABA).. Three groups of each biomaterial were evaluated: GA-treated, DE-treated and DE + CEABA-treated. The microstructure of non-implanted biomaterials was assessed by light microscopy after Picro Mallory staining; the phosphorus content of the DE and DE + CEABA samples was assessed by atomic emission spectrometry. Samples were implanted subcutaneously into young rats for 10 and 60 days. The explant end-point included quantitative calcification assessment by atomic absorption spectrophotometry and light microscopy examination after von Kossa staining.. All GA-treated biomaterials had a high calcium-binding capacity (>100 μg/mg dry tissue). DE preservation decreased the vein wall and pericardium calcium content by 4- and 40-fold, respectively, but was ineffective for the aortic wall. The calculated CEABA content was almost equal in the vein wall and pericardium (17.7 and 18.5 μM/g) and slightly less in the aortic wall (15 μM/g) (P = 0.011). CEABA effectively reduced mineralization in the DE aortic wall and DE pericardium to 10.1 (7.8-21.1) and 0.95 (0.57-1.38) μg/mg but had no effect in the DE vein wall. Mineralization in the GA- and DE-treated aortic and vein walls was predominantly associated with elastin. CEABA modification decreased elastin calcification but did not block it completely.. Each xenogeneic material requires individual anticalcification strategy. DE + CEABA pretreatment demonstrates a high mineralization-blocking efficacy for the bovine pericardium and should be employed to further develop the paediatric pericardial conduit. Aortic wall calcification cannot be blocked completely using this strategy.

Topics: Animals; Aorta; Biocompatible Materials; Bioprosthesis; Cattle; Diphosphonates; Elastin; Epoxy Resins; Glutaral; Heart Valve Prosthesis; Jugular Veins; Pericardium; Prosthesis Design; Rats; Swine; Tissue Culture Techniques; Tissue Preservation; Vascular Calcification

Accelerated elastin degradation is an important pathogenic mechanism in chronic obstructive pulmonary disease (COPD) leading to irreversible lung function loss and cardiovascular comorbidities. The rate of elastin breakdown is a predictor of mortality in patients with COPD. Decelerating elastinolysis might be an attractive therapeutic target in this debilitating condition. Vascular calcification starts in the elastin network of the arterial wall and is enhanced in patients with COPD. Elastin calcification is accompanied by an upregulation of matrix metalloproteinase gene expression and consequently a shift in the elastase/anti-elastase balance towards degradation. Magnesium can be regarded as a natural calcium antagonist and has the proven ability to ameliorate vascular calcification. Furthermore, an animal study has suggested that magnesium deficiency promotes elastin degradation. I hypothesize that inhibiting elastin calcification by means of magnesium supplementation might counteract both vascular calcification and elastin degradation in COPD. This could potentially have a favorable impact on cardiovascular and respiratory related morbidity/mortality in patients with COPD.

Topics: Animals; Elastin; Humans; Magnesium; Magnesium Deficiency; Models, Biological; Proteolysis; Pulmonary Disease, Chronic Obstructive; Renal Insufficiency, Chronic; Vascular Calcification

Atherogenesis is a multifactorial pathologic process influenced by genetics and environmental factors such as diet, exercise, stress, and other exposures. Estrogen receptors (ER) are expressed in cells of the arterial wall, suggesting that estrogen receptor ligands (estradiol, natural and pharmacologic ligands) may directly affect arterial biology and atherogenesis. Ligand bound estrogen receptor alpha and beta (ERα, ERβ) can influence physiology through direct binding to estrogen response elements in the DNA, through interactions with other transcription factors such as NF-κB, or through rapid effects not dependent on gene expression changes but instead through non-nuclear membrane sites involving ERα, ERβ, or G-coupled protein ER (GPER1). Elucidation of potential direct effects of estrogens on the artery wall requires careful evaluation of arterial biologic responses to estrogens. We have developed a comprehensive approach to understand the mechanisms of estrogen action which employs histologic measures of the size and other characteristics of atherosclerotic lesions, immunohistochemical assessments of cellular composition, evaluation of chemical, molecular, and genomic changes in the arterial environment, and determination of the relationships between arterial estrogen receptor expression and atherogenesis. This approach can provide important insights into the mechanisms of action of estrogen and other mediators of atherogenesis.

Topics: Animals; Arteries; Atherosclerosis; Biomarkers; Collagen; Disease Models, Animal; Elastin; Estrogens; Frozen Sections; Gene Expression Regulation; Immunohistochemistry; Lipids; Paraffin Embedding; Plaque, Atherosclerotic; Receptors, Estrogen; Tissue Fixation; Vascular Calcification; Workflow

Degeneration of elastin plays a vital role in the pathology and progression of abdominal aortic aneurysm (AAA). Our previous study showed that pentagalloyl glucose (PGG), a core derivative of tannic acid, hinders the development of AAAs in a clinically relevant animal model when applied locally. In this study, we tested whether targeted nanoparticles (NPs) can deliver PGG to the site of an aneurysm and prevent aneurysmal growth by protecting elastin. PGG-loaded albumin NPs with a surface-conjugated elastin-specific antibody were prepared. Aneurysms were induced by calcium chloride-mediated injury to the abdominal aorta in rats. NPs were injected into the tail vein after 10 days of CaCl

Topics: Animals; Antibodies; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Calcium Chloride; Cells, Cultured; Cytoprotection; Disease Models, Animal; Drug Compounding; Drug Liberation; Elastic Tissue; Elastin; Hydrolyzable Tannins; Injections, Intravenous; Macrophages; Male; Matrix Metalloproteinases; Nanoparticles; Particle Size; Proteolysis; Rats, Sprague-Dawley; Solubility; Vascular Calcification

Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects.. Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth.. Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition.. Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression.

Topics: Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Calcium Chloride; Chemistry, Pharmaceutical; Disease Models, Animal; Disease Progression; Drug Carriers; Elastin; Immunoconjugates; Macrophages; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Nanoparticles; Phenylalanine; Polyesters; Proteolysis; Rats, Sprague-Dawley; RAW 264.7 Cells; Thiophenes; Time Factors; Vascular Calcification

The purpose of this study was to analyze the association between morphological characteristics of human carotid plaques and patient's sex, age, and history of neurological symptoms.. The study included 763 atherosclerotic plaques from patients treated surgically for carotid stenosis between 2004 and 2013. Histological analyses of carotid plaques were performed to assess the type of plaque (American Heart Association classification), the stability of the plaque, the extent of calcification, inflammation, and neovascularization, as well as the deposition of collagen and elastin. According to the scale of outcome measurement, logistic regression, ordinal regression, and multinomial regression analyses were applied. All results were adjusted for common risk factors of atherosclerosis.. Male sex was associated with more cellularity (odds ratio [OR], 1.56; P=0.003), more inflammatory infiltrates (OR, 1.75; P<0.001), and more neovascularization (OR, 1.47; P=0.010), but less calcification (OR, 0.78; P=0.090). Symptomatic patients were more likely to have a lower amount of elastin (OR, 0.71; P=0.057). Higher age was associated with increased calcification (OR, 1.23; P=0.009). Unstable plaques were found more frequently in symptomatic patients (OR, 1.60; 95% confidence interval, 1.14-2.25; P=0.007). A multinomial regression model revealed that age, sex, and history of neurological symptoms were significantly associated with specific plaque types (P=0.009, P<0.001, and P=0.017, respectively).. Plaque morphology differed between men and women and varied with age. Certain types of plaques (VI and VI/VII) as well as unstable plaques were significantly associated with a history of neurological symptoms. Thus, individual approaches (eg, in detection of plaque hemorrhage or thin fibrous caps) especially based on sex and age should be considered to identify patients at increased risk of stroke.

Topics: Age Factors; Aged; Aged, 80 and over; Carotid Stenosis; Cohort Studies; Collagen; Cross-Sectional Studies; Elastin; Endarterectomy, Carotid; Female; Humans; Inflammation; Logistic Models; Male; Middle Aged; Neovascularization, Pathologic; Plaque, Atherosclerotic; Sex Factors; Vascular Calcification

Vascular calcification increases the risk of cardiovascular mortality. We previously reported that expression of elastin decreases with progression of inorganic phosphorus (Pi)-induced vascular smooth muscle cell (VSMC) calcification. However, the regulatory mechanisms of elastin mRNA expression during vascular calcification remain unclear. MicroRNA-29 family members (miR-29a, b and c) are reported to mediate elastin mRNA expression. Therefore, we aimed to determine the effect of miR-29 on elastin expression and Pi-induced vascular calcification. Calcification of human VSMCs was induced by Pi and evaluated measuring calcium deposition. Pi stimulation promoted Ca deposition and suppressed elastin expression in VSMCs. Knockdown of elastin expression by shRNA also promoted Pi-induced VSMC calcification. Elastin pre-mRNA measurements indicated that Pi stimulation suppressed elastin expression without changing transcriptional activity. Conversely, Pi stimulation increased miR-29a and miR-29b expression. Inhibition of miR-29 recovered elastin expression and suppressed calcification in Pi-treated VSMCs. Furthermore, over-expression of miR-29b promoted Pi-induced VSMC calcification. RT-qPCR analysis showed knockdown of elastin expression in VSMCs induced expression of osteoblast-related genes, similar to Pi stimulation, and recovery of elastin expression by miR-29 inhibition reduced their expression. Our study shows that miR-29-mediated suppression of elastin expression in VSMCs plays a pivotal role in osteoblastic differentiation leading to vascular calcification.

Topics: Calcium; Cell Differentiation; Cell Line; Down-Regulation; Elastin; Gene Expression Regulation; Humans; MicroRNAs; Muscle, Smooth, Vascular; Osteoblasts; Phosphorus; Vascular Calcification

Matrix gla protein (MGP) is a potent inhibitor of extracellular matrix (ECM) mineralization. MGP-deficiency in humans leads to Keutel syndrome, a rare genetic disease hallmarked by abnormal soft tissue calcification. MGP-deficient (Mgp(-/-)) mice show progressive deposition of hydroxyapatite minerals in the arterial walls and die within 2 months of age. The mechanism of antimineralization function of MGP is not fully understood. We examined the progression of vascular calcification and expression of several chondrogenic/osteogenic markers in the thoracic aortas of Mgp(-/-) mice at various ages. Although cells with chondrocyte-like morphology have been reported in the calcified aorta, our gene expression data indicate that chondrogenic/osteogenic markers are not upregulated in the arteries prior to the initiation of calcification. Interestingly, arterial calcification in Mgp(-/-) mice appears first in the elastic laminae. Considering the known mineral scaffolding function of elastin (ELN), a major elastic lamina protein, we hypothesize that elastin content in the laminae is a critical determinant for arterial calcification in Mgp(-/-) mice. To investigate this, we performed micro-computed tomography (µCT) and histological analyses of the aortas of Mgp(-/-);Eln(+/-) mice and show that elastin haploinsufficiency significantly reduces arterial calcification in this strain. Our data suggest that MGP deficiency leads to alterations of vascular ECM that may in turn initiate arterial calcification.

Topics: Aging; Animals; Antigens, Differentiation; Aorta, Thoracic; Durapatite; Elastin; Mice; Mice, Knockout; Proteins; Vascular Calcification; X-Ray Microtomography

Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation.

Topics: Actins; Aggrecans; Alkaline Phosphatase; Animals; Aorta; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Chondrogenesis; Collagen Type II; Core Binding Factor Alpha 1 Subunit; Durapatite; Elastin; Myosin Heavy Chains; Osteoblasts; Osteocalcin; Osteogenesis; Rats; Tunica Intima; Tunica Media; Vascular Calcification

This study investigated the effects of hypertension on regional aortic biomechanical and structural properties in three rat models of vascular calcification: the hypertensive Lewis polycystic kidney (LPK; n = 13) model of chronic kidney disease, spontaneously hypertensive rats (SHRs; n = 12), and calcification in normotensive Lewis rats induced by vitamin D3 and nicotine (VDN; n = 8). Lewis and Wistar-Kyoto rats were controls. Thoracic and abdominal aortic stiffness parameters were assessed by tensile testing. In models where aortic stiffness differences compared with controls existed in both thoracic and abdominal segments, an additional cohort was quantified by histology for thoracic and abdominal aortic elastin, collagen, and calcification. LPK and VDN animals had higher thoracic breaking strain than control animals (P < 0.01 and P < 0.05, respectively) and lower energy absorption within the tensile curve of the abdominal aorta (P < 0.05). SHRs had a lower abdominal breaking stress than Wistar-Kyoto rats. LPK and VDN rats had more elastic lamellae fractures than control rats (P < 0.001), which were associated with calcium deposition (thoracic R = 0.37, P = 0.048; abdominal: R = 0.40, P = 0.046). LPK rats had higher nuclear density than control rats (P < 0.01), which was also evident in the thoracic but not abdominal aorta of VDN rats (P < 0.01). In LPK and VDN rats, but not in control rats, media thickness and cross-sectional area were at least 1.5-fold greater in thoracic than abdominal regions. The calcification models chronic kidney disease and induced calcification in normotension caused differences in regional aortic stiffness not seen in a genetic form of hypertension. Detrimental abdominal aortic remodeling but lower stiffness in the thoracic aorta with disease indicates possible compensatory mechanisms in the proximal aorta.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Biomechanical Phenomena; Cholecalciferol; Collagen; Disease Models, Animal; Elastin; Female; Hemodynamics; Hypertension; Male; Oxazines; Rats; Rats, Inbred Lew; Rats, Inbred SHR; Rats, Inbred WKY; Renal Insufficiency, Chronic; Tensile Strength; Vascular Calcification; Vascular Stiffness

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp(-/-) mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp(-/-) animals. Nonetheless, the Mgp(-/-);Tgm2(-/-) mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp(-/-) animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp(-/-) vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp(-/-);Tgm2(-/-) mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.

Topics: Animals; Aortic Diseases; Calcium-Binding Proteins; Complement Factor D; Elastin; Extracellular Matrix Proteins; GTP-Binding Proteins; Matrix Gla Protein; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Vascular Calcification

Vascular calcification is common in chronic kidney disease, where cardiovascular mortality remains the leading cause of death. Patients with kidney disease are often prescribed vitamin D receptor agonists (VDRAs) that confer a survival benefit, but the underlying mechanisms remain unclear. Here we tested two VDRAs in a mouse chronic kidney disease model where dietary phosphate loading induced aortic medial calcification. Mice were given intraperitoneal calcitriol or paricalcitol three times per week for 3 weeks. These treatments were associated with half of the aortic calcification compared to no therapy, and there was no difference between the two agents. In the setting of a high-phosphate diet, serum parathyroid hormone and calcium levels were not significantly altered by treatment. VDRA therapy was associated with increased serum and urine klotho levels, increased phosphaturia, correction of hyperphosphatemia, and lowering of serum fibroblast growth factor-23. There was no effect on elastin remodeling or inflammation; however, the expression of the anticalcification factor, osteopontin, in aortic medial cells was increased. Paricalcitol upregulated osteopontin secretion from mouse vascular smooth muscle cells in culture. Thus, klotho and osteopontin were upregulated by VDRA therapy in chronic kidney disease, independent of changes in serum parathyroid hormone and calcium.

Topics: Animals; Aorta; Aortic Diseases; Calcitriol; Calcium; Cells, Cultured; Diet; Disease Models, Animal; Elastin; Ergocalciferols; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glucuronidase; Injections, Intraperitoneal; Klotho Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteopontin; Parathyroid Hormone; Phosphates; Receptors, Calcitriol; Renal Insufficiency, Chronic; Time Factors; Up-Regulation; Vascular Calcification