The Role of p53 in Atherosclerosis

Although the role of the tumour suppressor gene p53 is well known in cancer, recent studies have highlighted a fundamental role for p53 in regulating cells in the advanced atherosclerotic plaque, the major cause of heart attacks and stroke. In particular, p53 is activated in the complex environment of the plaque, in part by DNA damage within the lesion, and regulates growth arrest, cell senescence and apoptosis of vascular smooth muscle cells (VSMCs). The role of endogenous p53 has been determined using p53 knockout in mice developing advanced atherosclerosis, using bone marrow transplant to separate effects on blood cells from vessel wall cells. These studies have produced apparently contradictory and surprising results. In particular, recent studies have identified a role for endogenous p53 in protection of VSMCs from apoptosis, trans-differentiation of bone marrow stromal cells into VSMCs in atherosclerosis, and altering the mode of cell death in the plaque.     

Apoptosis in human atherosclerotic plaques

Intimal cell death has been a recognized feature of advanced atherosclerotic disease. With the advent of DNA in situ end labelling and/or ultrastructural techniques, recent findings suggest that cells of an atheroma undergo programmed cell death or apoptosis. The pathophysiologic relevance of apoptosis in atherosclerotic disease is debatable. Apoptotic cell death may influence lesion progression and thus reduce overall plaque burden. Alternatively, apoptosis may prove a means of quenching the inflammation, converting cellular-rich lesions to so-called stable fibrous hypocellular plaques or conversely weaken the fibrous cap causing plaque rupture, a major cause of acute coronary syndromes. Apoptotic cells within plaques are typically macrophages, smooth muscle cells and T-cells and the frequency of death varies in the different regions of the lesion. The precise signalling pathways of apoptosis in plaques are unknown. There is however, some evidence that production of immune cytokines may promote apoptosis through activation of the Fas ligand-mediated death pathway. Genetic signals that regulate apoptosis in the atheroma, at least in smooth muscle cells, may involve the tumour suppressor genes p105 ^RB and p53. Further studies as to the relevance of apoptosis in acute coronary syndromes and potential mechanisms are emerging.     

Detection of apoptosis in tissue sections

During the last few years, detection of apoptosis has evolved from a predominantly morphological basis to the use of ever more specific techniques. The methods widely used to visualize DNA fragmentation in tissue sections are now supplemented by a variety of specific antisera against components of the cell death pathways. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other forms of DNA damage, and the distinction between different stages of the cell death process. In this overview, we will focus on recent technical advances in apoptosis detection covering improvements of in situ DNA fragmentation techniques, as well as pointing out some of the new tools available for the detection of apoptotic cells in tissue.     

Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.     

Mast cells in vulnerable coronary plaques: potential mechanisms linking mast cell activation to plaque erosion and rupture

PURPOSE OF REVIEW: A novel link between inflammation and acute coronary syndromes is emerging, in that infiltrating inflammatory cells may convert a clinically silent coronary plaque into a dangerous and potentially lethal plaque. The majority of acute atherothrombotic events now relate to erosion or rupture of such unstable plaques. Here we summarize the molecular mechanisms by which activated mast cells may contribute to plaque erosion or rupture. RECENT FINDINGS: In-vitro experiments have revealed a multitude of paracrine effects exerted by activated mast cells. By secreting heparin proteoglycans and chymase, activated mast cells efficiently inhibit the proliferation of smooth muscle cells in vitro, and reduce their ability to produce collagen by a transforming growth factor beta-dependent and -independent mechanism. Mast cell chymase and tryptase are capable of activating matrix metalloproteinases types 1 and 3, causing degradation of the extracellular matrix component, collagen, necessary for the stability of the plaque. Activated mast cells also secrete matrix metalloproteinases types 1 and 9 themselves. Furthermore, chymase induces SMC apoptosis by degrading fibronectin, a pericellular matrix component necessary for SMC adhesion and survival, with the subsequent disruption of focal adhesions and loss of outside-in survival signaling. By secreting chymase and tumour necrosis factor alpha, activated mast cells also induce endothelial cell apoptosis. SUMMARY: Locally activated mast cells may participate in the weakening of atherosclerotic plaques by secreting heparin proteoglycans, chymase, and cytokines, which affect the growth, function and death of arterial endothelial cells and smooth muscle cells, thereby predisposing to plaque erosion or rupture.     

Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques

Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs) in the fibrous tissue or inflammatory (namely foam) cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffin-embedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose) polymerase-1 [PARP-1] fragment); biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis) were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions): the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis. Their death may decrease the cell number in the lipid core and generate a possibly defective apoptotic clearance: the resulting release of matrix-degrading enzymes could contribute to weakening the fibrous cap and promote the plaque rupture with the risk of acute ischemic events, while increasing the thrombogenic pultaceous pool of the plaque core.     

Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation

An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. Biochemical and genetic analyses of apoptosis provide an increasingly detailed picture of the intracellular signaling pathways involved. Nevertheless, it remains to be determined whether apoptosis can become a clinically important approach to modulate plaque progression. In this review, we have outlined some of the most recent results concerning apoptosis in atherosclerosis with a special focus on oxidized lipids, inflammation and therapeutic regulation of the apoptotic cell death process.     

"Vulnerable plaques"--ticking of the time bomb

Atherosclerosis and its sequelae are one of the leading causes of morbidity and mortality, especially in the developed nations. Over the years, treatment protocols have changed with the changing understanding of the disease process. Inflammatory mechanisms have emerged as key players in the formation of the atherosclerotic plaque. For the majority of its life span, the plaque develops silently and only some exhibit overt clinical manifestations. The purpose of this review is to examine the inherent properties of some of these "vulnerable" or symptomatic plaques. Rupture of the plaque is related to the thickness of the fibrous cap overlying the necrotic lipid core. A thin cap is more likely to lead to rupture. Multiple factors broadly grouped as the "determinants of vulnerability" are responsible for directly or indirectly influencing the plaque dynamics. Apoptosis is considered an important underlying mechanism that contributes to plaque instability. Inflammatory reactions within the plaque trigger apoptosis by cell-cell contact and intra cellular death signaling. Once started, the apoptotic process affects all of the components that make up the plaque, including vascular smooth muscle cells, endothelial cells, and macrophages. Extensive research has identified many of the key cellular and molecular regulators that play a part in apoptosis within the atherosclerotic lesion. This information will help us to gain a better understanding of the underlying mechanisms at the cellular and molecular level and enable us to formulate better therapeutic strategies to combat this disease.     

Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication.     

Apoptotic cell death in atherosclerosis

PURPOSE OF REVIEW: Apoptosis is a critical regulator of homeostasis in many tissues, including the vasculature. Apoptosis in atherosclerotic lesions is triggered by inflammatory processes, both via cell-cell contact and by cytokines and oxidized lipids. Apoptosis of vascular smooth muscle cells, endothelial cells and macrophages may promote plaque growth and pro-coagulation and may induce rupture, the major consequence of atherosclerosis in humans. RECENT FINDINGS: Studies over the past year have clearly demonstrated the significance of cell death in atherosclerosis. Some of the key cellular, cytokine and molecular regulators that contribute to the apoptosis of cells within the atherosclerotic lesion have been identified and their mechanism of action elucidated. Other studies have shed some light on the identity of cells whose loss by apoptosis contributes to plaque instability. SUMMARY: The identification of which cell types undergo apoptosis within the atherosclerotic lesion, the extracellular factors that impinge on these cells, and the intracellular mechanisms that govern their demise have begun to be elucidated. This information is critical in the design of further in-vivo experiments such as the exploitation of animal models, and ultimately, in applying this knowledge to clinical practice.     

实验性动脉粥样斑块内泡沫细胞凋亡及葛根总黄酮的干预作用

目的:观察ApoE基因敲除(ApoE gene knock out,ApoE-/-)小鼠主动脉窦动脉粥样硬化斑块超微结构改变,泡沫细胞凋亡,探讨葛根总黄酮(Total Flavone of Radix Puerariae,TFRP)的干预作用。方法:将16只Apo E-/-小鼠随机分为模型组及TFRP干预组(85 mg/kg.d),每组8只,2只C57BL/6J小鼠作为空白对照,12周后处死,用酶法检测二组血清脂质含量,采用光学显微镜、电子显微镜观察As斑块形态及泡沫细胞凋亡。结果:(1)两组血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)水平未见显著性差异;(2)光镜观察:模型组可见成熟的As斑块形成,部分斑块脂质核心较大,偏心性,纤维帽较薄,纤维帽内有较多炎症细胞浸润,平滑肌细胞成分少,肩部较薄弱,呈不稳定斑块形成趋势,在斑块脂质核心内可见到较多凋亡细胞;而TFRP干预组未见不稳定斑块,斑块脂质核心小,平滑肌细胞数目较多,纤维帽较厚,斑块内凋亡细胞数明显少于模型组(p<0.05)。(3)电镜观察:模型组斑块内以平滑肌源性泡沫细胞多见,可见中晚期凋亡细胞,细胞外基质成分较少;与模型组相比,TFRP干预组以巨噬细胞源性泡沫细胞多见,可见少数早期凋亡细胞,细胞外胶原纤维明显增多。结论:模型组病变处于中晚期As病变,符合泡沫细胞凋亡特征;TFRP干预抑制了ApoE基因敲除小鼠主动脉窦As斑块内泡沫细胞凋亡。     

Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor

Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.     

Arachidonic acid-derived oxidation products initiate apoptosis in vascular smooth muscle cells

The mechanism of arachidonic acid (AA)-induced apoptosis in vascular smooth muscle cells (VSMCs) was studied in the A-10 rat aortic smooth muscle cell line. Treatment of serum-deprived VSMCs with 50 microM AA for 24 h resulted in a loss of cell viability. The apoptotic effect of AA was characterized by annexin V binding, sub-G1 population of cells, cell shrinkage and chromatin condensation. AA-induced VSMC death was attenuated by antioxidants alpha-tocopherol and glutathione, the hydrogen peroxide (H2O2) scavenger catalase and by serum proteins, albumin and gamma globulins. Moreover, the AA peroxidation products, 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(S)-HPETE, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) caused VSMC apoptosis. These data suggest an oxidative mechanism of AA-induced VSMC death. The apoptotic effect of AA was pH-dependent, being inhibited by extracellular alkalinization to pH 8.0. AA inhibited serum-stimulated cell cycle progression in quiescent cells, but not in proliferating cells. In conclusion, AA, through its oxidation products causes VSMC apoptosis. Antioxidants, by inhibiting VSMC apoptosis, may prevent consequent pathological events such as atherosclerotic plaque rupture.     

Apoptosis in the lung: induction, clearance and detection

Apoptosis and other forms of programmed cell death are important contributors to lung pathophysiology. In this brief review, we discuss some of the implications of finding apoptotic cells in the lung and methods for their detection. The balance between induction of apoptosis and the normally highly efficient clearance of such cells shows that these are highly dynamic processes and suggests that abnormalities of apoptotic cell clearance may be an alternative explanation for their detection. Because recognition of apoptotic cells by other lung cells has additional effects on inflammation, immunity, and tissue repair, local responses to the dying cells may also have important consequences in addition to the cell death itself.     

程序性细胞死亡及其激光FCM检测方法

程序性细胞死亡是细胞生理性死亡最常见的形式,是细胞对其周围环境信号的主动反应。在此过程中细胞发生一系列特征性的变化,一些基因表达参与或调控此过程。ＦＣＭ是检测研究程序性细胞死亡的重要手段。诱导肿瘤细胞发生程序性死亡是肿瘤治疗的新途径。程序性细胞死亡的检测对肿瘤治疗研究及其临床疗效判断都有其应用前景。     

Akt regulates the survival of vascular smooth muscle cells via inhibition of FoxO3a and GSK3

Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.     

Induction of p53-dependent and mitochondria-mediated cell death pathway by dengue virus infection of human and animal cells

Previous studies have suggested that cells undergo apoptosis in response to dengue virus infection. However, the potential significance of dengue virus-induced apoptosis and the pathways are still not clearly defined. In this study, comparative analysis of dengue virus-induced apoptosis in BHK, H1299, HUH-7 and Vero cell lines was carried out. We show here that infection of BHK, HUH-7 and Vero cell lines with dengue type 1 virus (DEN1V) induces cell death typical of apoptosis. Virus-induced cell death was assayed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, detection of oligonucleosomal DNA fragmentation, DNA content analysis and assay for the externalization of phosphatidylserine residues. Detailed study of dengue virus infection in HUH-7 cells showed activation of cell death via the mitochondrial pathway causing lowering of mitochondrial transmembrane potential (DeltaPsim) in HUH-7 cells. Interestingly, in the p53-deficient cell line, H1299, apoptosis was largely undetectable compared with the other cell lines used; suggesting that a p53- and mitochondria-mediated cell death pathway may play an important role in dengue virus-induced apoptosis.     

植物细胞凋亡的ELISA检测(英文)

本文利用ＴＵＮＥＬ方法在单个细胞水平上对苯胺灵诱导的玉米根尖细胞死亡进行了检测。结果表明,一定浓度的苯胺灵能诱导玉米根尖细胞发生主动性的细胞凋亡,具有细胞凋亡典型的形态和生化特征即细胞核浓缩并形成核碎片、染色质边缘化及ＤＮＡ特异片段化等（Ｆｉｇ．１）。首次利用ＥＬＩＳＡ方法在群体水平上对这种细胞凋亡进行了进一步检测。结果表明：动物细胞研究常用的ＥＬＩＳＡ方法同样也适合用于植物细胞凋亡的检测。在凋亡前期,随着凋亡过程的进行,细胞质中的ＯＤ值逐渐上井（Ｆｉｇ．２）。剂量实验表明,０．２ｍｇ／ｍＬ苯胺灵最适合于诱导细胞凋亡（Ｆｉｇ．３）。     

Nitric oxide induces apoptosis in a human colonic epithelial cell line, T84

Chronic inflammation is associated with inducible nitric oxide synthase expression in infiltrating and resident cells (epithelia, neurons) and an exaggerated release of nitric oxide. NO can induce apoptosis in macrophages and tumour cell lines. We investigated whether NO induced cell death in an epithelial (T84) cell fine via apoptosis. Culture T84 cells were exposed to a bolus of NO (40 or 80 muM) dissolved in Hank's balanced salt solution (HBSS) supplemented with 10% fetal calf serum (FCS). After incubation for 4 h at 37( degrees )C in 5% CO(2), cells were either stained for DNA fragmentation with the TdT-mediated dUTP-biotin nick end labelling (TUNEL) method, or cytosolic DNA fragments quantified by a cell death detection ELISA assay. Nitric oxide induced apoptosis in a dose-dependent manner which preceded frank cell death (failure to exclude Trypan blue). These data suggest that epithelial cell death may be NO dependent and via apoptosis, in states of gut inflammation.     

Identification of apoptotic cell death in distraction osteogenesis

The purpose of this experimental work was to investigate whether apoptosis contributes to tissue remodelling during distraction bone healing. In a rabbit model of mandibular distraction osteogenesis, we quantitatively analysed the extent of apoptotic cell death in relation to differently applied mechanical loadings. Apoptotic cells were identified by means of an in situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure and by electron microscopical examination for typical morphological features of programmed cell death. TUNEL-positive cells were frequently detected in samples distracted at higher strain magnitudes. Ultrastructurally, these apoptotic cells displayed a condensed chromatin and fragmented nuclei, while the continuity of their plasma membranes remained intact. Our results clearly indicated that the discontinuous traction of osteotomized mandibles induced enhanced apoptosis. In contrast to non-distracted samples and mandibles distracted at low strain magnitudes, in which only minimal evidence of apoptotic cell death was detected, the application of hyperphysiological strain magnitudes resulted in an increased apoptosis rate. Thus, mechanical loading seems to be a triggering factor for apoptotic changes in osteoblastic cells. These findings suggest a pathophysiological role of apoptotic cell death in the control of tissue integrity during distraction osteogenesis.