The LDLR deficient mouse as a model for aortic calcification and quantification by micro-computed tomography (original) (raw)
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American Journal of Physiology-heart and Circulatory Physiology, 2010
Recent epidemiologic studies have revealed the risk factors associated for vascular atherosclerosis, including the male sex, smoking, hypertension, and elevated serum cholesterol, similar to the risk factors associated with the development of AV stenosis. An increasing number of models of experimental hypercholesterolemia demonstrate features of atherosclerosis in the AV, which are similar to the early stages of vascular atherosclerotic lesions. Experimental and clinical studies demonstrate that the hypercholesterolemic AV develops an atherosclerotic lesion which is proliferative and expresses high levels of osteoblast bone markers which mineralize over time to form bone. Calcification, the end-stage process of the disease, is necessary to understand as a prognostic indicator in the modification of this cellular process before it is too late. In summary, these findings suggest that medical therapies may have a potential role in patients in the early stages of this disease process to slow the progression to severe aortic stenosis and to delay the timing of the need for surgery. The translation of these experimental studies to clinical practice will be important to understand the potential for medical therapy for this disease process.
Atherogenesis and vascular calcification in mice expressing the human LPA gene
Pathophysiology, 2004
Background: Lp(a) lipoprotein (Lp(a)) contains polymorphic glycoprotein, apolipoprotein(a) (apo(a)) and low density lipoprotein (LDL). The extensive homology between apo(a) and plasminogen is believed to contribute to the pathogenicity of apo(a), but the precise mechanisms by which Lp(a) participates in atherogenesis is still unknown. We used LPA-yeast artificial chromosome (LPA-YAC) transgenic mice with or without the human APOB (hAPOB) gene to study pathogenicity of apo(a)/Lp(a) and illucidate its role in regulation of serum lipid levels. Methods: Middle-aged (1-year-old) mice were fed a control (AIN-76), a high-cholesterol (HC) or a high-cholesterol/high-fat (HCHF) diet for 7 weeks. For the study of serum total apo(a) and lipid levels, mice were sampled prior to the experiment, at 2 weeks and at 7 weeks when the animals were sacrificed. Hearts with ascending aorta were fixed in formalin, embedded in gelatine and prepared for sections on a cryostat. Livers were washed in ice cold saline and submerged in RNAlater TM buffer and stored at −70 • C until mRNA analysis. Results: Wild type mice fed the control diet did not develop aortic lesions. Presence of the LPA gene was sufficient to induce development of aortic lesions, but neither coexpression of the hAPOB gene nor feeding the HC diet or the HCHF diet augmented the development of aortic lesions in LPA-YAC transgenic mice. On the control diet transgenic females had larger aortic lesion size than transgenic males. Furthermore, aortic lesions in transgenic females were associated with calcification more often than in transgenic males. Serum total cholesterol levels were higher both in wild type and LPA-YAC transgenic males than in females mainly because of higher serum high-density lipoprotein cholesterol levels. HC and HCHF feeding had more pronounced effect on total cholesterol levels in LPA-YAC/hAPOB transgenic mice than in either wild type or LPA-YAC transgenic mice, due to increased low density lipoprotein cholesterol levels. Furthermore, these diets reduced serum total apo(a) levels in both transgenic mouse lines. Conclusion: Expression of the human LPA gene in mice is sufficient to trigger development of aortic lesions. Similar frequency of calcified lesions in LPA-YAC transgenic mice with or without hAPOB gene may suggest that apo(a) is the part of the Lp(a) molecule that causes aortic calcification. The basis for reduced serum total apo(a) level in response to cholesterol feeding is not clear, but interplay between LPA and factors involved in cholesterol or bile acid homeostasis is worth of future studies.
High-Density Lipoprotein Regulates Calcification of Vascular Cells
Circulation Research, 2002
Accumulating evidence has suggested the protective role of HDL in cardiovascular disease processes. Calcification is a common feature of atherosclerotic lesions and contributes to cardiovascular complications due to the loss of aortic resilience and function. Recent studies have suggested that vascular calcification shares several features with skeletal bone formation at the cellular and molecular levels. These include the presence of osteoblast-like calcifying vascular cells in the artery wall that undergo osteoblastic differentiation and calcification in vitro. We hypothesized that HDL may also protect against vascular calcification by regulating the osteogenic activity of these calcifying vascular cells. When treated with HDL, alkaline phosphatase activity, a marker of osteogenic differentiation of osteoblastic cells, was significantly reduced in those cells. Prolonged treatment with HDL also inhibited calcification of these cells, further supporting the antiosteogenic differentiation property of HDL when applied to vascular cells. Furthermore, HDL inhibited the osteogenic activity that was induced by inflammatory cytokines interleukin (IL)-1 and IL-6 as well as by minimally oxidized LDL. HDL also partially inhibited the IL-6-induced activation of signal transducer and activator of transcription 3 in calcifying vascular cells, suggesting that HDL may inhibit cytokine-induced signal transduction pathways. The inhibitory effects of HDL were mimicked by lipids extracted from HDL but not by HDL-associated apolipoproteins or reconstituted HDL. Furthermore, oxidation of HDL rendered it pro-osteogenic. Taken together, these results suggest that HDL regulates the osteoblastic differentiation and calcification of vascular cells and that vascular calcification may be another target of HDL action in the artery wall.
Cardiovascular Pathology, 2013
Background-Vascular calcification is highly prevalent in patients with type II diabetes mellitus (T2DM). Little is known about whether T2DM is causative. Methods-Low density lipoprotein receptor mutant (LDLr−/−) mice were fed with customized diabetogenic and/or procalcific diets to induce atherosclerosis, cartilaginous metaplasia and calcification, along with obesity, hyperglycemia, hyperinsulinemia, and hypercholesterolemia at various levels, and euthanized for study after 18-24 weeks on diet. Results-We found that T2DM accelerated cartilaginous and calcific lesion development by ~3and 13-folds as determined by incidence of vascular cartilaginous metaplasia and calcification in LDLr−/− mice. Lowering dietary fat from ~60% to ~40% kcal reduced body weight and serum glucose and insulin levels, leading to a 2-fold decrease in aortic calcium content. Correlation analysis of calcium content with a calculated insulin resistance index, HOMA-IR, showed a positive correlation of insulin resistance with vascular calcification. Finally, we used genetic fate mapping strategy to trace cells of SM origin in these animals. Vascular SMCs were found to be a major cell source contributing to osteochondrogenic differentiation and calcification. Receptor for advanced glycation end-products (RAGE) was up-regulated, co-localizing with osteochondrogenic SMCs. Conclusions-Through quantitative measure of aortic calcium content, we provided experimental findings that LDLr−/− mice, like T2DM patients, are predisposed to vascular calcification. Our study is also the first to establish a distinct role of hyperglycemia and hypercholesterolemia in osteochondrogenic differentiation of SMCs and determined these cells as a major source contributing to cartilaginous and calcifying lesions of T2DM blood vessels, possibly mediated by RAGE.
The lipid theory in the pathogenesis of calcific aortic stenosis
Nutrition, metabolism, and cardiovascular diseases : NMCD, 2015
Biologically active phenomena, triggered by atherogenesis and inflammation, lead to aortic valve (AV) calcification. Lipids play an important role in activating the cell signaling leading to AV bone deposition. This review, based on evidence from animal and human studies, mainly focused on the involvement of lipids and atherogenic phenomena in the pathogenesis of calcific aortic stenosis (AS). The role of elevated low density lipoproteins for the risk of both vascular atherosclerosis and AS has been elucidated. Lipid disorders act synergistically with other risk factors to increase prevalence of calcific AS. Atherosclerosis is also involved in the pathogenesis of bone demineralization, a typical hallmark of aging, which is associated with ectopic calcification at vascular and valvular levels. Animal studies have recently contributed to demonstrate that lipids play an important role in AS pathogenesis through the activation of molecular cell signalings, such as Wnt/Lrp5 and RANK/RANK...
Role of Cellular Cholesterol Metabolism in Vascular Cell Calcification
Journal of Biological Chemistry, 2011
Vascular calcification impairs vessel compliance and increases the risk of cardiovascular events. We found previously that liver X receptor agonists, which regulate intracellular cholesterol homeostasis, augment PKA agonist-or high phosphateinduced osteogenic differentiation of vascular smooth muscle cells. Because cholesterol is an integral component of the matrix vesicles that nucleate calcium mineral, we examined the role of cellular cholesterol metabolism in vascular cell mineralization. The results showed that vascular smooth muscle cells isolated from LDL receptor null (Ldlr ؊/؊) mice, which have impaired cholesterol uptake, had lower levels of intracellular cholesterol and less osteogenic differentiation, as indicated by alkaline phosphatase activity and matrix mineralization, compared with WT cells. PKA activation with forskolin acutely induced genes that promote cholesterol uptake (LDL receptor) and biosynthesis (HMG-CoA reductase). In WT cells, inhibition of cholesterol uptake by lipoprotein-deficient serum attenuated forskolin-induced matrix mineralization, which was partially reversed by the addition of cell-permeable cholesterol. Prolonged activation of both uptake and biosynthesis pathways by cotreatment with a liver X receptor agonist further augmented forskolin-induced matrix mineralization. Inhibition of either cholesterol uptake, using Ldlr ؊/؊ cells, or of cholesterol biosynthesis, using mevastatin-treated WT cells, failed to inhibit matrix mineralization due to up-regulation of the respective compensatory pathway. Inhibition of both pathways simultaneously using mevastatintreated Ldlr ؊/؊ cells did inhibit forskolin-induced matrix mineralization. Altogether, the results suggest that up-regulation of cholesterol metabolism is essential for matrix mineralization by vascular cells.
Regulatory role of endothelium in the expression of genes affecting arterial calcification
Biochemical and Biophysical Research Communications, 2004
Vascular calcification is a highly regulated process sharing features of bone mineralization. Since endothelium regulates many of the processes during atherogenesis, we monitored the expression of genes involved in calcification upon exposure of human coronary artery endothelial cells (HCAECs) to atherogenic stimuli. Genes studied were: core binding factor a-1 (Cbfa1/Runx2), a pivotal transcriptional regulator of osteogenesis; bone morphogenetic protein-2 (BMP2), an inducer of cartilage and bone; and matrix glaprotein (MGP), a potent inhibitor of calcification, which exerts its action by blocking BMP2. HCAECs were treated with oxidizedlow density lipoprotein (ox-LDL, 80 lg/mL) or tumor necrosis factor-a (TNFa, 10 ng/mL), and the expression of Cbfa1, BMP2, and MGP was quantified by real-time PCR. Cbfa1 was expressed at low levels in untreated HCAECs, and its expression did not change with ox-LDL or TNFa treatment. The expression of BMP2 and MGP increased early after exposure to ox-LDL or TNFa (at 2-8 h), and the increase was not evident at 24 h. Ox-LDL exerted a stronger effect on MGP than on BMP2 expression. The effects of ox-LDL, but not TNFa, on MGP and BMP2 expression were inhibited by pretreatment of cells with an antibody directed at LOX-1, a lectin-like receptor for ox-LDL (10 lg/mL). Thus, the endothelium, when exposed to atherogenic stimuli, ox-LDL in particular, regulates the process of calcification by enhancing the expression of the bone inhibitory MGP, while the expression of Cbfa1 remains unchanged. Upregulation of BMP2 may represent a feedback upregulation in response to increase in MGP. The effect of ox-LDL appears to be mediated by LOX-1 activation.
PLoS ONE, 2013
Background: There are no rigorously confirmed effective medical therapies for calcific aortic stenosis. Hypercholesterolemic Ldlr 2/2 Apob 100/100 mice develop calcific aortic stenosis and valvular cardiomyopathy in old age. Osteoprotegerin (OPG) modulates calcification in bone and blood vessels, but its effect on valve calcification and valve function is not known. Objectives: To determine the impact of pharmacologic treatment with OPG upon aortic valve calcification and valve function in aortic stenosis-prone hypercholesterolemic Ldlr 2/2 Apob 100/100 mice. Methods: Young Ldlr 2/2 Apob 100/100 mice (age 2 months) were fed a Western diet and received exogenous OPG or vehicle (N = 12 each) 3 times per week, until age 8 months. After echocardiographic evaluation of valve function, the aortic valve was evaluated histologically. Older Ldlr 2/2 Apob 100/100 mice were fed a Western diet beginning at age 2 months. OPG or vehicle (N = 12 each) was administered from 6 to 12 months of age, followed by echocardiographic evaluation of valve function, followed by histologic evaluation. Results: In Young Ldlr 2/2 Apob 100/100 mice, OPG significantly attenuated osteogenic transformation in the aortic valve, but did not affect lipid accumulation. In Older Ldlr 2/2 Apob 100/100 mice, OPG attenuated accumulation of the osteoblast-specific matrix protein osteocalcin by ,80%, and attenuated aortic valve calcification by , 70%. OPG also attenuated impairment of aortic valve function. Conclusions: OPG attenuates pro-calcific processes in the aortic valve, and protects against impairment of aortic valve function in hypercholesterolemic aortic stenosis-prone Ldlr 2/2 Apob 100/100 mice.