动脉粥样硬化斑块钙化机制的研究进展

心血管疾病中动脉粥样硬化斑块的钙化是动脉粥样硬化的临床标志之一,主要发生在动脉血管的内膜.动脉粥样硬化斑块核心的钙化不会增加斑块的易损性,而粥样斑块纤维帽上的微钙化会加强纤维帽的周向应力,致使斑块的易损性增加.动脉粥样硬化斑块的钙化机制包括被动钙化和主动钙化,被动钙化受激素和局部信号的调节,主动钙化机制涉及复杂的细胞生命过程,基质囊泡、细胞凋亡、外泌体、氧化应激反应和细胞自噬等均参与了钙化过程.本文对动脉粥样硬化斑块的钙化机制的进展进行综述.     

Human carotid plaque phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity,and enhances its uptake by macrophage at the expense of its binding to HDL

Human carotid atherosclerotic plaque is in direct contact with circulatory blood components. Thus, plaque and blood components may affect each other. The current study presents the effects of plaque chloroform:methanol (C:M) extract on the HDL-associated enzyme paraoxnase 1 (PON1). This study is part of our investigation on the mutual effects of the interactions between atherosclerotic lesions and blood components. Recombinant PON1 (rePON1) was incubated with the human carotid plaques C:M extract and PON1 activities were analyzed. Lactonase and paraoxonase activities were elevated due to C:M treatment, by 140 and by 69%, respectively. Analytical chemistry analyses revealed specific phosphatidylcholines (PCs) as the plaque active components. Tryptophan fluorescence quenching assay, together with molecular docking, shows that PON1 activity is enhanced in correlation with the level of PC affinity to PON1. Molecular docking revealed that PCs interact specifically with H2-PON1 α-helix, which together with H1 enzyme α-helix links the protein to the HDL surface. These findings are supported by additional results from the PON1 ∆20 mutant that lack its H1-α-helix. Incubation of this mutant with the plaque C:M extract increased PON1 activity by only 20%, much less than the wild-type PON1 that elevated PON1 activity at the same concentration by as much as 95%. Furthermore, as much as the affinity of the enzyme to the PC was augmented, the ability of PON1 to bind to the HDL particle decreased. Finally, PON1 interaction with PC enhance its uptake into the macrophage cytoplasm. In conclusions, Specific lesion phosphatidylcholines (PCs) present in the human carotid plaque significantly enhance PON1 catalytic activities due to their interaction with the enzyme. Such a lesion׳s PC–PON1 interaction, in turn, competes with HDL PCs and enhances PON1 uptake by macrophage at the expense of PON1 binding to the HDL.     

A gene score of nine LDL and HDL regulating genes is associated with fluvastatin-induced cholesterol changes in women

While conventional pharmacogenetic studies have considered single gene effects, we tested if a genetic score of nine LDL- and HDL-associated single nucleotide polymorphisms, previously shown to predict cardiovascular disease, is related to fluvastatin-induced lipid change. In patients with asymptomatic plaque in the right carotid artery, thus candidates for statin therapy, we related score LDL [APOB(rs693), APOE(rs4420638), HMGCR(rs12654264), LDLR(rs1529729), and PCSK9(rs11591147)] and score HDL [ABCA1(rs3890182), CETP(rs1800775), LIPC(rs1800588), and LPL(rs328)] as well as the combined score LDL+HDL to fluvastatin-induced LDL reduction (± metoprolol) (n = 395) and HDL increase (n = 187) following 1 year of fluvastatin treatment. In women, an increasing number of unfavorable alleles (i.e., alleles conferring higher LDL and lower HDL) of score LDL+HDL (P = 0.037) and of score LDL (P = 0.023) was associated with less pronounced fluvastatin-induced LDL reduction. Furthermore, in women, both score LDL+HDL (P = 0.001) and score HDL (P = 0.022) were directly correlated with more pronounced fluvastatin-induced HDL increase, explaining 5.9–11.6% of the variance in treatment response in women. There were no such associations in men. This suggests that a gene score based on variation in nine different LDL- and HDL-associated genes is of importance for the magnitude of fluvastatin HDL increase in women with asymptomatic plaque in the carotid artery.     

Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.     

Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation

Paraoxonase 1, an HDL-associated enzyme that confers antioxidant activity on HDL, and its activity in serum have been correlated with protection against atherosclerosis, an oxidative disease. However, serum PON-1 activity is highly variable and its regulation is complex, involving both genetic and environmental factors. It is influenced by gender and inflammation, two important factors in atherosclerosis. Serum PON-1 activity has been shown to be lower in male mice and is decreased in male Syrian hamster during inflammation. Here we show that male mice had lower hepatic PON-1 mRNA that increased by 170% after castration. Our data also suggested that this effect was testes but not plasma testosterone dependent. Ovariectomy had no effect on PON-1 mRNA in female mice. LPS caused hepatic PON-1 mRNA to decrease further in male mice, and to increase moderately in female mice. Anti-inflammatory dexamethasone enhanced PON-1 mRNA level by 2-fold in male and female LPS-treated mice, and increased PON-1 expression by 8-fold in Hepa cell, a mouse hepatoma cell line. Therefore, antioxidant PON-1 is regulated at the mRNA level in a gender-specific manner by proinflammatory LPS and anti-inflammatory dexamethasone.     

Protein deglycase DJ-1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability

Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE^−/− mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe^−/− mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe^−/−DJ-1^−/− mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe^−/−DJ-1^+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.     

Tankyrase-1 function at telomeres and during mitosis is regulated by Polo-like kinase-1-mediated phosphorylation

Telomere length is critical for chromosome stability that affects cell proliferation and survival. Telomere elongation by telomerase is inhibited by the telomeric protein, TRF1. Tankyrase-1 (TNKS1) poly(ADP-ribosyl)ates TRF1 and releases TRF1 from telomeres, thereby allowing access of telomerase to the telomeres. TNKS1-mediated poly(ADP-ribosyl)ation also appears to be crucial for regulating the mitotic cell cycle. In searching for proteins that interact with polo-like kinase-1 (Plk1) by using complex proteomics, we identified TNKS1 as a novel Plk1-binding protein. Here, we report that Plk1 forms a complex with TNKS1 in vitro and in vivo, and phosphorylates TNKS1. Phosphorylation of TNKS1 by Plk1 appears to increase TNKS1 stability and telomeric poly(ADP-ribose) polymerase (PARP) activity. By contrast, targeted inhibition of Plk1 or mutation of phosphorylation sites decreased the stability and PARP activity of TNKS1, leading to distort mitotic spindle-pole assembly and telomeric ends. Taken together, our results provide evidence of a novel molecular mechanism in which phosphorylation of TNKS1 by Plk1 may help regulate mitotic spindle assembly and promote telomeric chromatin maintenance.     

Aging of mesenchymal stem cells is a root cause of vascular calcification

We hypothesize that aging of mesenchymal stem cells breaking the balance between arterial mineral deposition and resorption results in vascular calcification. It is believed that it is a new idea about revealing pathogenesis of artery calcification and defining a range of novel and exciting therapeutic approaches.     

Expression of nuclear transport importins beta 1 and beta 3 is regulated during rodent spermatogenesis

Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.     

Mechanical action of the blood onto atheromatous plaques: influence of the stenosis shape and morphology

The vulnerability of atheromatous plaques in the carotid artery may be related to several factors, the most important being the degree of severity of the endoluminal stenosis and the thickness of the fibrous cap. It has recently been shown that the plaque length can also affect the mechanical response significantly. However, in their study on the effect of the plaque length, the authors did not consider the variations of the plaque morphology and the shape irregularities that may exist independently of the plaque length. These aspects are developed in this paper. The mechanical interactions between the blood flow and an atheromatous plaque are studied through a numerical model considering fluid–structure interaction. The simulation is achieved using the arbitrary Lagrangian–Eulerian scheme in the COMSOL TM commercial finite element package. The stenosis severity and the plaque length are, respectively, set to 45% and 15 mm. Different shapes of the stenosis are modelled, considering irregularities made of several bumps over the plaque. The resulting flow patterns, wall shear stresses, plaque deformations and stresses in the fibrous cap reveal that the effects of the blood flow are amplified if the slope upstream stenosis is steep or if the plaque morphology is irregular with bumps. More specifically, the maximum stress in the fibrous cap is 50% larger for a steep slope than for a gentle slope. These results offer new perspectives for considering the shape of plaques in the evaluation of the vulnerability.