Atherosclerosis: Pathophysiology of insulin resistance, hyperglycemia, hyperlipidemia, and inflammation

Journal of DiabetesVolume 12, Issue 2 p. 102-104 COMMENTARYFree Access Atherosclerosis: Pathophysiology of insulin resistance, hyperglycemia, hyperlipidemia, and inflammation 动脉粥样硬化:胰岛素抵抗、高血糖、高血脂及炎症的病理生理学 Joshua K. Beverly, Joshua K. Beverly Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CaliforniaSearch for more papers by this authorMatthew J. Budoff, Corresponding Author Matthew J. Budoff mbudoff@labiomed.org Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, California Correspondence Matthew Budoff, Los Angeles Biomedical Research Institute at Harbor-UCLA, 1124 W Carson Street, Torrance CA 90502. Email: mbudoff@labiomed.orgSearch for more papers by this author Joshua K. Beverly, Joshua K. Beverly Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CaliforniaSearch for more papers by this authorMatthew J. Budoff, Corresponding Author Matthew J. Budoff mbudoff@labiomed.org Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, California Correspondence Matthew Budoff, Los Angeles Biomedical Research Institute at Harbor-UCLA, 1124 W Carson Street, Torrance CA 90502. Email: mbudoff@labiomed.orgSearch for more papers by this author First published: 14 August 2019 https://doi.org/10.1111/1753-0407.12970Citations: 41AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Atherosclerotic cardiovascular disease (CVD) is on course to surpass infectious diseases as the leading cause of morbidity and mortality worldwide.1 Multiple risk factors are responsible for this trend including the increasing average life expectancy and reducing rates of communicable diseases in addition to potentially modifiable risk factors, such as tobacco use, hypertension, hyperlipidemia, and diabetes mellitus.1 The development of atherosclerosis is driven by multiple factors including hypertension, dyslipidemia, inflammation, insulin resistance, and hyperglycemia. While many techniques are available to accurately measure atherosclerosis (coronary artery calcium scanning, CT angiography, intravascular ultrasound), understanding the pathophysiology of the disease may help drive discovery to ultimately prevent the disease. 1 INSULIN RESISTANCE AND ATHEROSCLEROSIS An important distinction to draw is the role of insulin resistance vs hyperglycemia in the development of atherosclerosis. Although both likely have a synergistic atherogenic effect in the setting of type 2 diabetes, insulin resistance has been shown to have a strong link to CVD, even in the absence of hyperglycemia.2 Insulin resistance promotes a pro-inflammatory state and dyslipidemia in addition to perturbed insulin signaling on important intimal cells (endothelial, vascular smooth muscle cells [SMCs], and macrophages) resulting with advanced plaque progression in the setting of hyperinsulinemia.2 Normal insulin signaling in skeletal muscle starts with insulin binding to its receptor, which causes tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) to exert insulin's effect on glucose metabolism.2 Meanwhile in the liver, IRS-1/IRS-2 activates phosphatidylinositol 3 (Pl-3)–kinase, which phosphorylates PI, PI-4, and PI-4,5, and mediates glucose transport and glycogen synthase.3 in vivo trials conducted in hyperinsulemic, euglycemic lean type II diabetics and obese nondiabetics have shown that the impaired phosphorylation of IRS-1 and PI-3–kinase activation caused significant dysfunction in glucose and glycogen synthesis.4 Nitric oxide synthase is activated through the same PI-3–kinase pathway; the resultant decrease in nitric oxide production leads to endothelial dysfunction and accelerated atherosclerosis.4 2 HYPERGLYCEMIA AND ATHEROSCLEROSIS Chronic hyperglycemia has been shown to interfere with multiple metabolic pathways resulting in microvascular complications (eg, retinopathy, neuropathy, and nephropathy).5 However, there is uncertainty in the literature on the degree of impact hyperglycemia exerts on macrovascular outcomes compared to other risk factors.6 Nevertheless, recent meta-analyses have found that elevated blood glucose is an independent risk factor for cardiovascular and all-cause mortality in both diabetic and nondiabetic patients.7 Three major mechanisms have been described to facilitate these outcomes: (a) nonenzymatic glycosylation of proteins and lipids, (b) oxidative stress, and (c) protein kinase C (PKC) activation.8 Advanced glycation end products (AGEs) occur through the Maillard reaction in which reducing sugars undergo nonenzymatic reactions leading to the formation of reactive carbonyl compounds and the subsequent glycooxidation of proteins, lipids, and nucleic acids.9 AGEs accumulate with advancing age, but this process markedly increases in the setting of hyperglycemia, oxidative stress, and inflammation.9 The mechanisms in which AGEs further atherosclerosis can be classified as nonreceptor dependent and receptor mediated (Aronson). The prototypical nonreceptor mechanism is the alteration in the normal physiology of the low-density lipoprotein (LDL) particle.8 Glycosylation of the apoprotein B (Apo B) and phospholipid components of LDL result in disturbances in LDL clearance and susceptibility to oxidative modification.8 Human monocyte-derived macrophages have a higher affinity for glycated LDL via the nonspecific (scavenger) receptor which stimulates foam cell formation and promotes atherosclerosis.8 Furthermore, glycation of LDL confers increased susceptibility to LDL becoming oxidized, which is a key step in atherogenicity.8 Aminoguanidine is an inhibitor of AGE formation, but has not been clinically efficacious due to toxicity limitations.5 The receptor-mediated mechanism involves the binding of AGE receptor (RAGE).8 RAGE has been demonstrated in all cells relevant to atherosclerosis including monocyte-derived macrophages, endothelial cells, and SMCs.8 Oxygen-free radicals leading to atherosclerosis and diabetic complications is a long-held theory.5 Glucose is metabolized into reductive equivalents which drive the generation of adenosine triphosphate via oxidative phosphorylation with free radicals as byproducts.5 “Increased oxidant stress reduces nitric oxide levels, damages cellular proteins, and promotes leukocyte adhesion to the endothelium while inhibiting its barrier function.”5 These effects ultimately result in accelerated atherosclerosis. PKCs are chronically elevated in diabetics. The physiologic activation of PKC occurs from activating phospholipase C and increasing calcium and diacylglycerol (DAG) levels, which in turn activate PKC.5 In diabetes, pathologic activation can occur through elevation of glyceraldehyde-3-phosphate, subsequent elevation of DAG, and ultimately, activation of PKC.5 PKC pathway dysfunction can have many downstream consequences, including increased permeability, nitric oxide dysregulation, increased leukocyte adhesion, and induction of growth factor expression (vascular endothelial growth factor [VEGF], transforming growth factor beta [TGF-beta]).5 3 CHOLESTEROL AND ATHEROSCLEROSIS An undeniable causal relationship exist between plasma cholesterol levels and atherosclerosis.1 Atherogenesis is mediated in large part by the endothelium causing inflammation and accumulation of oxidatively modified LDL in the intima of the vessel wall facilitating monocyte recruitment and foam cell formation.10 Typically, the endothelium acts as a selective barrier between blood and tissues with increased permeability at arterial branch points/curvatures.10 The initial, most atherogenic event is accumulation of LDL in the subendothelial matrix.10 Conditions for this event are optimal when circulating amounts of LDL are increased and high-density lipoprotein (HDL; a lipid which transports excess cholesterol from peripheral tissues for storage/degradation in the liver) is decreased.10 LDL is taken up by macrophages to form foam cells once it is sufficiently oxidized by a multiplicity of factors including reactive oxygen species, myeloperoxidase, sphingomyelinase, and a secretory phospholipase (group II sPLA2).10 Macrophages primarily uptake the LDL by scavenger receptors SR-A and CD 36.10 4 INFLAMMATION AND ATHEROSCLEROSIS Historically, the pathogenesis of atherosclerosis was based on the lipid theory with explanations related to excess cholesterol being the sole cause of lipid deposition in the arterial wall. In the past two decades, it has been increasingly recognized that inflammation is involved in every step of the atherosclerotic process.11, 12 Currently, atherogenesis is characterized by chronic accumulation of monocytes/macrophages, SMCs, and lymphocytes which release pro-inflammatory molecules within the arterial wall.11, 12 As a result, ongoing research is being performed in order to create anti-inflammatory therapies to prevent the development of atherosclerotic plaques.11 Both innate and adaptive responses of the immune system are leveraged in atherogenesis. As mentioned previously, the initial event for atherogenesis is lipid retention in the intima of arteries enabled by endothelial dysfunction from insult-induced damage. Next, the trapped LDL is modified by enzymes and oxygen radicals, which subsequently stimulates endothelial cells to express adhesion molecules (eg, vascular cell adhesion molecule 1 [VCAM-1] and intercellular adhesion molecule 1 [ICAM-1]) and vascular SMCs to release chemokines (CCR2, ZCCR5, ZCX3CR1, and their ligands) and chemoattractants, which recruit “inflammatory” monocytes and T cells into the developing plaque.11, 13 Monocytes differentiate in situ into macrophages and uptake oxidized LDL. The adaptive immune system begins to facilitate atherogenesis in the early stages as well when lymphocytes transmigrate into the arterial wall.11 Helper T (TH) cells have multiple subtypes; most notably, TH1 cells are clearly atherogenic, whereas regulatory T cells (Tregs) are atheroprotective; the other subtypes do not yet have clearly defined roles.11 The protein component of the LDL particle is presented by macrophages and dendritic cells to T lymphocytes via the major histocompatibility complex class II (MHC-II), and the T cells produce pro-inflammatory cytokines.11 Another key component of the adaptive response is B cells. B1 cells predominantly produce IgM antibodies, which are protective of atherosclerosis, while B2 cells produce IgG antibodies, which promote atherosclerosis by interacting with CD4 T cell activation and stimulating effector T cell proliferation.11 Toll-like receptors play a central role in innate and adaptive immune responses and appear to be expressed in atherogenic leukocytes, including monocytes/macrophages, dendritic cells, and T and B lymphocytes.11 As the inflammatory process becomes chronic, SMCs also start to migrate into the intimal layer of the artery in response to chemokines, aided by release of matrix metalloproteinases (MMPs).12 With chronicity, plaque vulnerability becomes the primary determinant of thrombus- and rupture-mediated complications.13 Macrophages become pivotal in plaque destabilization through various enzymes, bioactive mediators; but notably, MMP has been implicated as an important pathogenic molecule contributing to plaque destabilization.13 Eventually, continuous apoptosis of macrophages and accumulation of nondegradable necrotic debris leads to the formation of the lipid-laden necrotic core seen in late lesions.13 Fibrous plaques are characterized by a growing mass of extracellular lipid and by accumulation of SMCs and SMC-derived extracellular matrix.10 The most vulnerable plaques to rupture generally have thin fibrous caps and increased numbers of inflammatory cells.10 5 CONCLUDING REMARKS Atherosclerosis is a chronic disease which only stands to become even more significant as life expectancy continues to increase and risk factors, such as hypertension, diabetes, tobacco use, and hyperlipidemia become more prevalent. We have demonstrated multiple etiologies and accompanying mechanisms contributing to the formation and propagation of atherosclerotic plaques.14, 15 Going forward, it will be important that our understanding of this complex disease continues to evolve in concert with imaging modalities and novel therapeutic targets to detect and prevent the disease. ACKNOWLEDGEMENT No funding received. DISCLOSURE Dr Budoff reports grant support from the National Institutes of Health and General Electric. No other author reports a conflict. 动脉粥样硬化性心血管疾病(cardiovascular disease，CVD)正在超过感染性疾病，成为全球发病率和死亡率的首要原因。除了吸烟、高血压、高脂血症以及糖尿病等可改变的风险因素之外，还有多种其他风险因素导致了这一趋势，包括平均预期寿命的延长和感染性疾病发病率的降低1。 动脉粥样硬化的发生是由高血压、血脂异常、炎症、胰岛素抵抗、高血糖等多种因素驱动的。虽然目前有许多技术可用于准确测量动脉粥样硬化(如冠状动脉钙化扫描、CT血管造影、血管内超声)，但是了解疾病的病理生理学可能有助于更早地发现并最终预防该疾病。 1 胰岛素抵抗与动脉粥样硬化 一个重要区别是胰岛素抵抗与高血糖在动脉粥样硬化形成中的作用。尽管二者在2型糖尿病患者中可能具有协同致动脉粥样硬化作用，但已证实即使在没有高血糖的情况下，胰岛素抵抗与CVD之间仍具有很强的相关性2。 在高胰岛素血症的情况下，胰岛素抵抗除了扰乱重要内膜细胞(内皮细胞、血管平滑肌细胞[smooth muscle cells，SMCs]与巨噬细胞)的胰岛素信号外，还促进促炎症状态与血脂异常，导致动脉斑块进展2。在骨骼肌中正常的胰岛素信号传导始于胰岛素与其受体结合，使胰岛素受体底物1(insulin receptor substrate 1，IRS-1)中的酪氨酸磷酸化，从而发挥胰岛素对葡萄糖代谢的作用2。与此同时，在肝脏中，IRS-1/IRS-2激活磷脂酰肌醇3(phosphatidylinositol 3，Pl-3)-激酶，使PI、PI-4与PI-4,5磷酸化，并介导葡萄糖转运与糖原合成酶3。在高胰岛素血症、血糖正常的瘦型2型糖尿病患者和肥胖的非糖尿病患者中进行的体内试验表明，IRS-1与PI-3-激酶激活磷酸化受损可导致葡萄糖与糖原合成出现显著障碍4。一氧化氮合酶通过相同的PI-3激酶途径激活；一氧化氮生成减少可导致内皮功能障碍，并加速动脉粥样硬化4。 2 高血糖与动脉粥样硬化 已证实慢性高血糖会干扰多种代谢途径，导致微血管并发症(如视网膜病变、神经病变和肾病)5。然而，与其他危险因素相比较，文献中高血糖对大血管结局的影响程度存在不确定性6。尽管如此，最近的meta分析发现，血糖升高是糖尿病与非糖尿病患者心血管和全因死亡的独立危险因素7。目前已描述了三种主要机制可导致这些结果:(a)蛋白质与脂质的非酶糖基化，(b)氧化应激和(c)蛋白激酶C(protein kinase C，PKC)激活8。 晚期糖基化终末产物(AGEs)通过美拉德反应发生，其中还原糖发生非酶促反应，导致活性羰基化合物的形成，以及随后蛋白质、脂质与核酸的糖氧化9。AGEs随着年龄的增长不断积累，但这一过程在高血糖、氧化应激以及炎症的情况下显著加快9。AGEs进一步致动脉粥样硬化的机制可分为非受体依赖性与受体介导性。 典型的非受体机制是低密度脂蛋白(LDL)颗粒的正常生理学变化8。载脂蛋白B(Apo B)与LDL磷脂组分的糖基化导致LDL清除紊乱，易发生氧化修饰8。人单核细胞来源的巨噬细胞通过非特异性(清道夫)受体对糖化LDL具有更高的亲和力，这会刺激泡沫细胞形成并促进动脉粥样硬化。此外，LDL糖基化可增加LDL氧化的易感性，这是动脉粥样硬化发生过程中的关键步骤8。氨基胍是一种AGE形成抑制剂，但由于毒性限制，尚无临床疗效5。 受体介导的机制涉及与AGE受体(RAGE)的结合8。目前已在所有与动脉粥样硬化相关的细胞(包括单核巨噬细胞、内皮细胞与平滑肌细胞)中证实存在RAGE8。 氧自由基可导致动脉粥样硬化与糖尿病并发症，这是一个由来已久的理论5。葡萄糖被代谢为还原当量，通过氧化磷酸化作用与作为副产物的自由基驱动三磷酸腺苷的生成5。“氧化应激增加可降低一氧化氮水平，损害细胞蛋白，促进白细胞粘附于内皮细胞，同时抑制其屏障功能”5。这些作用最终导致动脉粥样硬化加速。 糖尿病患者的PKC长期处于高水平。PKC的生理激活途径是通过激活磷脂酶C，增加钙与二酰甘油(diacylglycerol，DAG)水平，进而激活PKC5。在糖尿病患者中，病理性激活途径是通过甘油醛-3-磷酸水平升高，随后DAG水平升高，并最终激活PKC而发生的。PKC通路功能障碍可产生许多后果，包括通透性增加、一氧化氮失调、白细胞粘附增加和诱导生长因子表达(血管内皮生长因子[vascular endothelial growth factor，VEGF]，转化生长因子β[transforming growth factor beta，TGF-β])5。 3 胆固醇与动脉粥样硬化 血浆胆固醇水平与动脉粥样硬化之间存在不可否认的因果关系1。动脉粥样硬化形成在很大的程度上由内皮细胞介导，引起炎症和氧化修饰的 LDL 在血管壁内膜聚集，促进单核细胞募集与泡沫细胞形成10。通常，内皮作为血液和组织之间的选择性屏障，在动脉分支点/弯曲处的渗透性增加10。最初的致动脉粥样硬化事件是LDL在内皮下基质中的蓄积10。最容易发生这种事件的条件是循环中的LDL水平上升并且高密度脂蛋白(HDL；是一种将外周组织中多余的胆固醇运输到肝脏中进行储存/降解的脂蛋白)水平下降10。一旦LDL被多种因素(包括活性氧、髓过氧化物酶、鞘磷脂酶以及分泌型磷脂酶[II组sPLA2])充分氧化，就会被巨噬细胞摄取形成泡沫细胞10。巨噬细胞主要是通过清道夫受体SR-A与CD 36来摄取LDL10。 4 炎症与动脉粥样硬化 历来所阐释的动脉粥样硬化发病机制是基于脂质理论，认为过量的胆固醇是导致动脉壁脂质沉积的唯一原因。在过去的二十年里，人们越来越多地认识到，炎症参与了动脉粥样硬化的过程的每一个环节11,12。目前，动脉粥样硬化的特征是单核/巨噬细胞、平滑肌细胞和淋巴细胞在动脉壁内长期积聚，并释放促炎分子11,12。因此，目前正在开展研究来创建抗炎疗法，用于预防动脉粥样硬化斑块的发生11。免疫系统的固有反应和适应性反应对动脉粥样硬化的形成均有影响。 如前所述，动脉粥样硬化形成的起始事件是损伤引起的内皮功能障碍使得脂质滞留在动脉内膜。接着，被困的LDL经过酶与氧自由基的修饰，随后刺激内皮细胞表达粘附分子(如血管细胞粘附分子1[VCAM-1]与细胞间粘附分子1 [ICAM-1])和血管平滑肌细胞释放趋化因子(CCR2、ZCCR5、ZCX3CR1及其配体)与化学吸引剂，募集“炎性”单核细胞与T细胞进入正在形成的斑块中11,13。单核细胞原位分化为巨噬细胞并摄取氧化型LDL。 在早期阶段，当淋巴细胞进入动脉壁时，适应性免疫系统也开始促进动脉粥样硬化的形成11。辅助性T(TH)细胞有多种亚型；最值得注意的是，TH1细胞具有明确的致动脉粥样硬化作用，而调节性T细胞(Tregs)具有动脉粥样硬化保护作用；其他亚型的作用尚未明确11。LDL颗粒中的蛋白质成分由巨噬细胞与树突状细胞通过主要组织相容性复合体II(MHC-II)递呈给T淋巴细胞，随后T细胞产生促炎细胞因子11。适应性反应的另一个关键成分是B细胞。B1细胞主要产生可以预防动脉粥样硬化的IgM抗体，而B2细胞产生的IgG抗体通过与CD4 T细胞活化相互作用，并刺激效应T细胞增殖，促进动脉粥样硬化形成11。 Toll样受体在先天性与适应性免疫应答中发挥核心作用，并且似乎是在致动脉粥样硬化的白细胞中表达，包括单核/巨噬细胞、树突状细胞以及T和B淋巴细胞11。随着炎症过程转为慢性，在趋化因子的作用下，并在基质金属蛋白酶(MMPs)释放的辅助下，平滑肌细胞也开始迁移到动脉内膜层12。随着时间的推移，斑块易损性成为血栓与破裂介导并发症的主要决定因素13。巨噬细胞(通过多种酶和生物活性介质)在导致斑块不稳定中发挥关键作用；但是值得注意的是，MMP被认为是导致斑块不稳定的重要致病分子。最终，巨噬细胞的持续凋亡和不可降解的坏死碎片的积聚导致在晚期病变中形成富含脂质的坏死核心13。纤维斑块的特征是细胞外脂质的不断增多，以及平滑肌细胞和平滑肌细胞衍生的细胞外基质积聚10。最易破裂的斑块通常具有薄的纤维帽以及更多的炎性细胞10。 5 总结 动脉粥样硬化是一种慢性疾病，随着预期寿命的延长，危险因素(如高血压、糖尿病、吸烟以及高脂血症)变得更加普遍，这种疾病只会变得越来越显著。目前我们已经证实多种病因与相关机制可促进动脉粥样硬化斑块的形成和进展。展望未来，我们需要加深对这一复杂疾病的认识，不断改进影像学方法并发现新的治疗靶点，从而早期检测和预防这种疾病。 REFERENCES 1Levenson JW, Skerrett PJ, Gaziano JM. Reducing the global burden of cardiovascular disease: the role of risk factors. Preventative Cardiology. 2007; 5: 188- 199. 2Bornfeldt KE, Tabas I. Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metabolism Review. 2011; 14: 575- 585. 3Defronzo RA. Insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing links. The Claude Bernard lecture 2009. Diabetologia. 2010; 53(7): 1270- 1287. 4Pendergrass M, Bertoldo A, Bonadonna R, et al. Muscle glucose transport and phosphorylation in type 2 diabetic, obese nondiabetic, and genetically predisposed individuals. Am J Physiol Endocrinol Metab. 2007; 292(1): E92- E100. 5Sheetz MJ, King GL. Molecular understanding of hyperglycemia's adverse effects for diabetic complications. JAMA. 2002; 288(20): 2579- 2588. 6 Yurong. Glycosylated hemoglobin in relationship to cardiovascular outcomes and death in patients with type 2 diabetes: a systematic review and meta-analysis. PLoS One. 2012; 7(8):e42551. 7Cavero - Redondo I, Peleteiro B, Alvarez-Bueno C, Rodriguez-Artalejo F, Martinez-Vizcaino V. Glycated hemoglobin A1c as a risk factor of cardiovascular outcomes and all-cause mortality in diabetic and non-diabetic populations: a systematic review and met - analysis. BMJ Open. 2017; 7(7):e015949. 8Aronson, D, Rayfield, E. J. “How hyperglycemia promotes atherosclerosis: molecular mechanisms.” Cardiovasc Diabetol 2002; 1: 1– 10 9Fishman SL, Sonmez H, Basman C, Singh V, Poretsky L. The role of advanced glycation end-products in the development of coronary artery disease in patients with and without diabetes mellitus: a review. Mol Med. 2018; 24: 59. 10Lusis AJ. Atherosclerosis. Nature. 2000; 407(6801): 233- 241. 11Li B, Li W, Li X, Zhou H. Inflammation: a novel therapeutic target/direction in atherosclerosis. Curr Pharm Des. 2017; 23(8): 1216- 1227. 12Milioti N, Bermudez-Farjardo A, Penichet ML, Oviedo-Orta E. Antigen-induced immunomodulation in the pathogenesis of atherosclerosis. Clin Dev Immunol. 2008;723-729. 13Zhong-qun Y, Hansson GK. Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev. 2007; 219(1): 187- 203. 14Budoff M, Backlund JC, Bluemke DA, et al. The Association of Coronary Artery Calcification With Subsequent Incidence of Cardiovascular Disease in Type 1 Diabetes: The DCCT/EDIC Trials. JACC Cardiovasc Imaging. 2019; 12:1341-1349. 15Budoff MJ, Raggi P, Beller GA, et al. Imaging Council of the American College of cardiology. Noninvasive cardiovascular risk assessment of the asymptomatic diabetic patient: the Imaging Council of the American College of Cardiology. JACC Cardiovasc Imaging. 2016; 9(2): 176- 192. Citing Literature Volume12, Issue2February 2020Pages 102-104 ReferencesRelatedInformation