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Pathophysiology and biochemistry of cardiovascular disease
Pathophysiology and biochemistry of cardiovascular disease James Scott Atherosclerosis is the major cause of cardiovascular disease. Hypercholesterolaemia, hypertension and cigarette smoking are the common risk factors for atherosclerosis. These risk factors unite behind a convergence of mechanism, involving oxidation and inflammation in the artery wall that, with time, gives rise to characteristic fatty-fibrous lesions. Physical trauma and inflammation produce lesion rupture, which can lead to clinical events such as heart attack and stroke, or resolve with plaque growth. Disease progression is marked by the inflammatory indicator CRP (C-reactive protein). Early indicators of heart attack are the inflammatory marker CD40, and the cardiac myofilament protein troponin. Coronary atherosclerosis is the common cause of heart failure (HF). Disordered calcium signalling to the myofilaments occurs in HF and in cardiomyopathy. Enhanced calcium signalling suppresses HF. Neuro-humoral and biomechanical processes, as seen in hypertension, produce cardiac hypertrophy, which predisposes to HF through apoptosis. Although in humans cardiac damage produces permanent loss of cells, because the heart cannot regenerate, developments in stem cell technology suggest that help is at hand.
Introduction Cardiovascular disease, including heart attack, stroke and heart failure (HF), is the leading cause of disease and death in the developed world, and is poised to become the most significant health problem worldwide. Atherosclerosis is the major cause of cardiovascular disease. Table 1 shows the risk factors for atherosclerosis and the main patho-biological processes on which they converge. This review concentrates on the pathogenesis of atherosclerosis as a disease driven by oxidative stress and enhanced inflammation in the artery wall. The role of inflammation in lesion progression and in current and emerging treatment measures is described. New markers of disease activity and myocardial infarction are described. Atherosclerosis is also the driver of the emerging epidemic of HF. The role of abnormal calcium handling in this disorder is described along with factors that lead to cardiac hypertrophy and HF as well as the potential of stem cell therapy for repairing the damaged heart.
Pathogenesis of atherosclerosis
Current Opinion in Genetics & Development 2004, 14:271–279
Autopsy studies show that atherosclerosis develops slowly over many years. A similar disease is produced in experimental animals, where a high-cholesterol diet and genetic manipulation cause accelerated lesions. These studies have produced a picture of the evolution of atherosclerosis from fatty streak to the complex plaque and clinical disease (Figure 1).
This review comes from a themed issue on Genetics of disease
Oxidative processes
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0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.04.012
Abbreviations CM cardiomyopathy CRP C-reactive protein DCM dilated cardiomyopathy ER endoplasmic reticulum GPCR G-protein-coupled receptor HB-EGF heparin-binding epidermal growth factor HCM hypertrophic cardiomyopathy HDL high-density lipoprotein HF heart failure LDL low-density lipoprotein MMPs matrix metalloproteases PLN phospholamban PPAR peroxisome proliferator activated receptor SERCA2a SR Ca2þ-ATP SMC smooth muscle cell SR sarcoplasmic reticulum TLRs Toll-like receptors www.sciencedirect.com
LDL and other nutrient macromolecules are transported in caveolae through endothelial cells into the vascular intima. LDL is retained in the sub-endothelium by a charged interaction with matrix proteoglycans [1]. Trapped LDL is attacked and oxidised by reactive oxygen species (superoxide anion, hydrogen peroxide, lipid peroxides and peroxynitrite), of which ozone is the latest and most malignant member of the ‘family’ [2]. Reactive oxygen species are inactivated by dietary antioxidants and enzymes such as glutathionine peroxidase, the principal cellular anti-oxidant enzyme [3]. Low levels of this enzyme predispose to atherosclerosis. High oxidative stress is also found in hypertension (mediated through abnormal renal salt handling and increased production of angiotensin II) and cigarette smoking (as a result of reduced glutathione peroxidase) [3,4]. This provides a compelling convergence of mechanisms for the major risk factors for atherosclerosis (Table 1). Inflammation and lesion formation
Oxidized LDL is engorged by macrophages, which take on a characteristic foamy appearance, and become Current Opinion in Genetics & Development 2004, 14:271–279
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Table 1 Risk factors for atherosclerosis. Risk factors
Primary pathogenetic process
Not modifiable Age Ethnicity Male gender Genetics Modifiable by lifestyle change Diet high in saturated fat and cholesterol, low in fruit, vegetables and grain Obesity Smoking Lack of exercise Modifiable by drugs Dyslipidaemia Hypertension Non-traditional Lipoprotein (a) Homocysteine Infection Systemic lupus erytyematosis
recruited and induced to proliferate and secrete collagen by growth factors such as PDGF and TGF-b. In time, the characteristic fatty-fibrous lesion of atherosclerosis develops (Figure 1). Lesion progression
High blood cholesterol, oxidative stress Metabolic syndrome of insulin resistance Oxidative stress Poor perfusion, adverse lipid profile Oxidative stress Oxidative stress, enhanced vasoconstriction Thrombogenesis Oxidative stress Inflammation Inflammation
activated leading to a state of chronic inflammation (Figure 1) [5]. Cytokines (IL-1, IFN-g, TNF-b, and angiotensin II) and chemokines (MCP-1, IL-8, IFN-g, CXC cytokines, and eotaxin) promote adhesion molecule expression (VCAM-1, P- and E-selectin) and attract immune and inflammatory cells (monocytes, leukocytes, B and T lymphocytes, and mast cells), mainly in regions of turbulent blood flow. Leukocyte attachment to adhesion molecules causes them to move out of the blood stream and roll along the surface of endothelial cells. They egress from the blood at endothelial cell tight junctions (Figure 1). Smooth muscle cells (SMCs) are
The growth of the atheromatous plaque is not a continuous process. Lesions undergo extensive remodelling, and neo-vascularisation, punctuated by bursts of activity and regression, which may produce cardiac events. The stable plaque has a preponderance of SMCs embedded in a dense matrix of collagen. Such plaques are typically achieved by lipid-lowering therapy. They are low in inflammatory cell content and cytokines such as bFGF, VEGF and PDGF promote endothelial cell and SMC survival [6]. The unstable plaque usually has a large core of inflammatory cells and debris from dead cells. The cap of these plaques is often fragile and denuded of endothelial cells. These lesions are subject to excessive inflammation. Inflammatory cells produce pro-apoptotic cytokines, such as TNF-b, for SMC and endothelial cells, matrix metalloproteases (MMPs), which digest collagen fibrils, and cytokines, such as IFN-g, which inhibit collagen secretion from SMCs. Cholesterol accumulation in macrophages signals death by perturbing the ER membranes, which depletes Ca2þ stores and induces an unfolding protein response, leading to activation of the cell death effector CHOP [7]. These characteristics make such plaques particularly prone to physical disruption. Although plaque rupture is the cause of clinical events, in many cases it is silent (i.e. cause no clinical symptoms). In such silent events, the thrombus generated is absorbed into the existing plaque. Plaque growth is then stimulated by the release from platelets of PDGF and TGF-b, which together stimulate SMC recruitment and proliferation and the production of a dense collagen matrix. Remodelling is also mediated through autocrine effects of PDGF and
(Figure 1 Legend) Progressive cellular and molecular mechanisms in the origins of atherosclerotic plaque, and common processes involved in hypertrophy, dilatation and heart failure. (a) The three diagrams show the development and progression of atherosclerotic lesions from fatty streak, to fibrous plaque and ruptured plaque. LDL is trapped in the sub-endothelial space, where it undergoes oxidation. Monocytes attach to endothelial cells and migrate into the intima through tight junctions. This process involves an intimate interaction with the junctional adhesion molecule PECAM and the release of spare membrane from cellular invaginations [38]. Monocytes become activated, and express the scavenger receptors CD36 and SR-A, which promote the uptake of oxidised LDL. Homeostatic responses facilitate reverse cholesterol transport through HDL. Macrophages signal the recruitment and activation of T-lymphocytes, the two cells together perform the concert of activity that leads to atherosclerosis. This involves SMC, controlled by interaction between PDGFR (platelet derived growth factor receptor) and LRP (low density lipoprotein related protein [39], recruitment and proliferation and production of the dense plaque matrix [39]. Pro-inflammatory and pro-apoptotic processes and physical disruption pre-dispose to plaque rupture. Macrophage secretion of MMPs and microvascularisation contribute to plaque weakness and susceptibility to rupture. Plaque rupture exposes blood components to tissue factor initiating coagulation and the recruitment of platelets. Formation of thrombus may lead to catastrophic clinical events or resolution by incorporation into plaque. After Glass and Witztum [40]. (b) The principal causes of cardiac hypertrophy are listed. Hypertrophic cardiomyopathy (HCM) is the common cause of sudden death in adolescents and young adults, especially in athletes [41]. It is an autosomal dominant disorder affecting sarcomeric proteins, which leads to disruption of force transmission and cellular hyperplasia, and hypertrophy of the ventricles. Cardiac function deteriorates as myocytes undergo apoptosis, followed by scar formation leading to sudden death or heart failure. The principal causes of cardiac dilatation are listed. Dilated cardiomyopathy (DCM) is the most frequent form of cardiomyopathy. It occurs mainly in children. DCM has both acquired and genetic causes. Adenovirus or coxsacchie virus infection can progress to autoimmune destruction of the myocardium and expansion of the ventricles. Inherited defects of a variety of proteins can cause similar changes in the ventricles. These include several sarcomeric proteins, also associated with HCM [41]. Current Opinion in Genetics & Development 2004, 14:271–279
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Pathophysiology and biochemistry of cardiovascular disease Scott 273
Figure 1
(b)
(a) Vascular lumen
Adhesion VCAM-1 P-selectin E-selectin ICAM-1 PECAM
Monocyte
Endothelial cells
MCP-1 CCR-2 IL-8 CXC
Extracellular matrix
Intima
Lymphocytes Migration LDL HDL LDL oxidation
Differentiation M-CSF
mmLDL
15 LO INOS Ozone
MMPs
Homeostatic responses
Tissue factor
apo E ABC A1 ACAT
Cytokines
Foam cell
ROS
ox LDL
Cardiac hypertrophy
Internal elastic lamina ox LDL uptake
Hypertension Hypertrophic cardiomyopathy
scaveger receptors CD36 SR-A
Media
Macrophage
Smooth muscle cells
Vascular lumen Endothelial cells Fibrinogen
Fibrous cap Fibrin Collagen Proteoglycans
Cytokines PDGF TGF-β
IL-4 IL-10
Macrophage foam cells
IL-1 IFN-γ Th2
PDGFR LRP
TNF-β CD40
IL-4 IL-10 Th1
Smooth muscle cells
Coronary arteries
Vascular lumen
Cardiac dilatation and heart failure
Platelets SMC-derived foam cells CD40 PDGF
Necrotic core CD40
Tissue factor
Atherosclerosis Hypertension Metabolic syndrome Dilated cardiomyopathy
Extracellular lipid 'Gruel'
Macrophage foam cell
MMPs
IFN-γ Th1 cell Current Opinion in Genetics & Development
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274 Genetics of disease
New micro-vessels in the plaque are fragile and likely to rupture, generating intra-plaque haemorrhage, which may resolve contributing further to plaque growth, or accrete cholesterol from red cell membranes, which contribute to plaque instability [10].
which is suppressed by PPARa and g agonists. PPARd, another member of the family, enhances fat burning in fatty tissue and muscle [11,12]. It also represses macrophage activity by a novel mechanism in which the binding of ligand to the nuclear receptor PPARd leads to release of BCL-6, a transcriptional repressor. In the absence of ligand, BCL-6 remains bound to PPARd resulting in a pro-inflammatory response.
Management of atherosclerosis
Oxidation and inflammation
Established treatment
For many non-traditional risk factors, the strength of evidence supporting their role in atherogenesis is not strong, perhaps because their role is either modest or complex (Table 1). For example, oxidative stress is a pervasive unifying mechanism in atherogenesis, but clinical trials using anti-oxidants show uniformly negative results. However, a recent large prospective population study showing the benefits of traditional Mediterranean diet, and its presumed suppression of oxidative stress, suggests that the situation is more complex, and that a varied diet provides the necessary anti-oxidants, which simple supplementation does not [13].
TGF-b, which operate under the control of the retinoicacid receptor and Kru¨ ppel-like transcription factors [8,9].
Reduction of elevated levels of circulating LDL cholesterol is the best treatment we have for atherosclerosis. However, LDL lowering decreases the risk of cardiac events by only 30%, even with optimum treatment of other major risk factors. This is perhaps because disease is well established by the time treatment is started. Moreover, target ‘normal’ cholesterol levels are probably high for our species, as compared to human neonates and animals in the wild. Elucidation of atherogenic mechanisms, particularly those involved in oxidation and inflammation, and better evaluation of non-traditional risk factors such as lipoprotein (a) and infection, may in the future lead to improved therapy (Table 1). Some recent insights from genetics are shown in Table 2. Emerging risk factors
A prominent and serious risk factor is the metabolic syndrome of insulin resistance (Table 1), which is driven by obesity. Insulin resistance leads to diabetes, with the generation of highly atherogenic advanced glycation end products, an aggressive atherogenic lipoprotein profile (hypertriglyceridaemia, reduced HDL [high-density lipoprotein], and readily oxidised, small dense LDL), hypertension, and a pro-coagulant state. Adipocytes in the obese have many parallels with macrophage foam cells. In the obese, macrophages also pervade fatty tissue. Both cell types engage in lipid storage and cytokine secretion,
Inflammation is now seen as so central to atherogenesis that it is an obvious therapeutic target [5]. Indeed, the drugs used for the treatment of atherosclerosis — aspirin, statins, PPARa agonists and angiotensin II signalling inhibitors — all have anti-inflammatory effects [5,14]. However, the use of broad-spectrum anti-inflammatory or immunosuppressive drugs for the treatment of a chronic disease like atherosclerosis compromises host defences. The successful treatment of cardiac transplant vasculopathy illustrates this. Transplant vasculopathy is a relentlessly progressive diffuse SMC proliferation. It is treated successfully with a new class of macrocyclic immunosuppressive agents with unique anti-proliferative activity, but this success is compromised by a high rate of bacterial infection (see also Table 2: TLR4, genetic variation,
Table 2 Genetic studies and atherosclerosis. Gene defect
Clinical syndrome, molecular mechanism
Refs
(a)
Atherosclerosis, increased leukotrienes, increased inflammation recruitment of T-cells and macrophages, increased vascular permeability enhanced by dietary arachidonic acid Common stroke, atherosclerosis, degradation cAMP, immune modulation Myocardial infarction, cytokine induction of adhesion molecules Myocardial infarction, gap junction between endothelial cells Myocardial infarction, fibrinolysis Myocardial infarction, MMP Myocardial infarction, proliferation of SMCs, co-operates with GATA1 to control the expression of atrial natriuretic factor Myocardial infarction gene not identified Atherosclerosis decreased, innate immune response to gram negative organism decreased, susceptibility to infection increased Augmented response of HDL cholesterol to hormone replacement therapy
[42] [43,44] [45] [46] [47] [47] [47] [48]
5-lipoxygenase activation 5-lipoxygenase activating protein (b) Phosphodiesterase 4D (c) Lymphotoxin-a (c) Connexin 37 (c) Plasminogen activator inhibitor 1 (c) Stromelysin (b) Myocyte enhancer factor 2 (b)
(b) (a)
Linkage chromosome 14 Toll-like receptor 4
(a)
Estrogen Receptor
(a)
(b)
Candidate gene;
linkage analysis;
(c)
[49] [50] [51]
large-scale association study.
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Pathophysiology and biochemistry of cardiovascular disease Scott 275
infection and atherosclerosis) [15]. Nonetheless, the macrocyclic agents are highly successful for short-term, local use in the prevention of endothelial cell and SMC proliferation after stenting at the time of coronary angioplasty [16]. In consideration of possible new therapeutic targets, the redundancy of distal effectors of inflammation suggest that narrow-spectrum distal inhibition will not modify disease effectively. Rather, the targeting of proximal triggers, such as ozonolysis [2], may prove a more promising strategy for interrupting inflammation in atherogenesis [5]. Infection
A variety of studies link acute respiratory infections and organisms such as Chlamydia pneumonae, Helicobacter pylori and cytomeglalovirus to atherosclerosis, but the role of pathogens remains unclear. Recent studies make an important link [17]. Bacterial lipopolylsaccharides are cleared by Toll-like receptors (TLRs), which are crucial for defence against infection. TLR stimulation releases antimicrobial peptides, cytokines, and initiates adaptive immunity. TLRs also block the activation of the nuclear receptor LXR (liver X nuclear receptor). This decreases the activity of the ABC1 transporter and pre-disposes macrophages to foam cell formation (see also Table 2: genetic variation, infection and atherosclerosis). Infection is also considered as a link to the general vascular inflammation that accompanies unstable angina [18]. Markers of atherosclerosis and myocardial infarction
C-reactive protein (CRP) is an important marker of general inflammation and of atherosclerosis [19]. It is independent of other markers such as LDL. CRP is produced by the liver in response to cytokines such as IL-6. It binds LDL/ oxidised LDL and is present in lesions, where it may act as a chemoattractant, and be involved in the expression of adhesion molecules. Its role as a marker for predictive screening of atherosclerosis, which contributes to other markers, remains to be established in randomised clinical trials. Whether markers such as products of ozonolysis will predict atherosclerosis requires clarification [2]. The bulk of cardiac troponin T forms an important component of the myocardial sarcomere (see Calcium handling in disease, below); the remainder is free in the cytoplasm [20]. When myocardial damage occurs, the cytoplasmic pool is released, followed by the protracted release of myofilament tropinin. Troponin is a much more sensitive measure of cardiac damage than the conventional MB isoenzyme fraction of myocardial creatine kinase. Cardiac troponin levels in the blood predict short-term prognosis in acute coronary syndromes particularly in microinfarction. Soluble CD40 ligand, a member of the TNF family, is another predictor of acute coronary syndromes [21,22]. Soluble CD40 ligand is expressed on leukocytes, www.sciencedirect.com
endothelial cells, smooth muscle cells and activated platelets. Although not a general marker of atherosclerosis, it appears to predict acute coronary syndromes and is independent of troponin. Elevation of soluble CD40 ligand indicates platelet activation and increased risk of cardiovascular events. Patients with elevated CD40 levels should benefit from anti-platelet treatment.
The heart HF has for the patient a prognosis worse than many forms of cancer [23,24]. Coronary atherosclerosis, hypertension and the metabolic syndrome are the major causes of HF. The primary cardiomyopathies (CMs) and arrhythmias are rare, but have received considerable attention because of their often dramatic presentation with sudden death, increased frequency in children, and the important insights they provide into common HF (Figure 1). Normal calcium handling
A frequent common feature of HF, CM and cardiac arrhythmia is abnormal myocyte Ca2þ signalling. Normal cardiac contractions are initiated by the entry of Ca2þ into cells. This leads to the release of large amounts of Ca2þ required for muscle contraction from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR/RyR2). A member of the immunophilin family of cytosolic binding proteins, FK506 binding protein (FKBP12/12.6), stabilises RYR preventing aberrant activation of the channel during the resting phase of the cardiac cycles. During exercise, RYR phosphorylation by protein kinase A dissociates FKPB12/12.6 from the channel increasing Ca2þ release and cardiac contractility. Variation in Ca2þ concentration is transferred to the thin sacromeric filaments (actin, tropomycin and troponin) by the Ca2þ receptor troponin. Three genes encode the troponin subunits for Ca2þ binding (TnC), inhibition (TnI), and tropomysin-binding (TnT). The structure of troponin in the Ca2þ-saturated form provides important insights into the role of troponin in the initiation of contraction. At low intracellular Ca2þ concentration, TnI interacts with actin and blocks the actomyosin ATPase. Increased Ca2þ binding to TnC releases TnI from its interaction with actin so that inhibition is released and contraction initiated. Cardiac contraction ends when Ca2þ is pumped back into the SR. This is regulated by the protein phospholamban (PLN), which in its unphosphorylated form sits on and inhibits the SR Ca2þ-ATP (SERCA2a) pump. Phosphorylation of PLN, in response to signals such as adrenaline, detaches PLN from the pump, allowing Ca2þ to move into the SR more quickly, and causing the heart to beat more efficiently. Calcium handling in disease
Mutations in the genes encoding Naþ and Kþ channels involved in cardiac action potentials underlie fatal cardiac arrhythmias in inherited long QT syndrome. Defects in proteins involved in Ca2þ transport also cause fatal arrhythmia. Mutation of ankryn B causes long QT Current Opinion in Genetics & Development 2004, 14:271–279
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syndrome and arrhythmia in humans and mice, by disruption of the cellular organisation of the Naþ pump, the Naþ/Ca2þ exchanger and the inositol-1, 4, 5-tryphosphate receptors (all ankryn B binding proteins) [25]. Mutations in the RyR2 Ca2þ channel also trigger fatal cardiac arrhythmias. Recent studies help clarify the link between acute infection and chronic autoimmune injury, and abnormal Ca2þ handling in both primary and acquired dilated cardiomyopathy (DCM). The autoimmune induction of antibodies against cardiac TnI, which is also defective in primary DCM, and the administration of monoclonal antibodies to TnI induce DCM in wild-type mice [26,27]. Antibodies against TnI augment the voltage-dependent L-type calcium current of cardiomyocytes, suggesting that antibodies to TnI induce DCM by chronic stimulation of calcium influx in cardiomyocytes. Mutation of the PLN gene leads to DCM with refractory HF. Cellular and biochemical studies indicate the mutant PLN, unlike wild-type, does not inhibit SERCA2a directly (Figure 1) [24]. This suggests that therapies aimed at preventing PLN from inhibiting the SERCA2a pump might be used to treat ordinary HF. Indeed HF progression in DCM is suppressed by a pseudo-phosphorylated mutant of a PLN via in vivo cardiac rAAV gene delivery [28]. In line with these findings in mice, patients with idiopathic DCM, who improve on b blocker treatment, show increases in SERCA2a and a-myocin heavy chain mRNAs and a decrease in b-myocin heavy chain mRNA, changes consistent with our understanding of the pathogenesis of disease, and the role of Ca2þ handling in this [29]. Cardiac hypertrophy and heart failure
Pressure overload in hypertension is triggered by two types of input that result in cardiac hypertrophy: neurohumoral mediators that activate growth factor receptor tyrosine kinases, cytokine receptors and G-proteincoupled receptors (GPCRs); and biomechanical stress. Stimulation of adrenergic receptors results in activation of G proteins, which can signal by triggering the synthesis of cAMP. This leads to the activation of Ca2þ regulatory proteins (including the plasma membrane Ca2þ channel and PLN) and myofilament proteins. This, in turn, leads to altered sarcomeric protein synthesis and cardiac hypertrophy, which may be beneficial as it allows the heart to work harder. However, it may also reduce the efficiency of structural adaptation leading to dilatation and HF. Double homozygotes for gain of function genetic variation of the b1 and a2C-adrenergic receptors produces substantial increased risk of HF in patients [30,31]. To clarify the mechanism of these processes, a mouse model of the b1 receptor variant has been generated. Importantly the mice, as do humans with this SNP, show Current Opinion in Genetics & Development 2004, 14:271–279
enhanced b-blocker responsiveness and reduced cardiac deterioration, thus indicating a preventative treatment strategy. GPCR signalling is now linked to cardiac hypertrophy. GPCR signalling can also operate through phospholipase C, which generates diacylglycerol. Diacylglycerol is a potent activator of protein kinase C, whose association with a distintergin metalloprotein called ADAM12 leads to the shedding of membrane-bound heparin-binding epidermal growth factor (HB-EGF) [32]. HB-EGF is then available to transactivate the EGF receptor, ErbB1, which stimulates cellular growth leading to cardiac hypertrophy. By contrast, the antibody herceptin (used in the treatment of breast cancer) that blocks the ErbB2 receptor gives rise to CM. The generation of mice with ventricle-restricted deletion of ErbB2 also show CM. Together these results indicate that ErbB1 and -2 signalling determine the balance between cardiac hypertrophy and cardiac dilatation. The role of biomechanical sensors in cardiac hypertrophy is less well understood than neuro-hormonal mechanisms. Transduction of mechanical stress into biochemical signals is mediated largely by integrins that couple the extracellular matrix to the cellular cytoskeleton. An important protein that acts as a biomechanical sensor in this pathway is melusin, which interacts with the cytoplasmic domain of the integrins, and is expressed only in skeletal and cardiac muscle [33]. Mice deficient for melusin still show cardiac hypertrophy in response to hormones acting through GPCRs, but not in response to biomechanical stress. However, in response to biomechanical pressure overload, melusin-deficient mice develop DCM and HF. These results indicate that melusin protects against HF, probably activating protective pathways that allow cardiac hypertrophy. The loss of cardiomyocytes through apoptosis is a key event in the development of HF. The molecular mechanisms that induce cell death have now been elucidated in pressure overload cardiac hypertrophy by gene-expression profiling. The mitochondrial death protein Nix is induced and triggers apoptotic cardiomyopathy in Gqmediated pressure overload cardiac hypertrophy [34]. These data establish a molecular mechanism for induction of cardiomyocyte apoptosis in hypertrophied myocardium and for its progression to HF. Repair of the damaged heart
Amphibians and zebrafish can regenerate heart tissue but mammals cannot [35,36]. Thus, after myocardial infarction, and in CM and HF there is permanent cell loss. A remedy for this would be to induce the heart to make new cells by the division of existing cells. One strain of mouse can apparently regenerate and a series of genes have been identified that allow this. Considerable debate exists as to www.sciencedirect.com
Pathophysiology and biochemistry of cardiovascular disease Scott 277
whether circulating or resident stem cells can differentiate into mature cardiac tissue in humans. A recent study suggests a stem cell population exists in the adult heart and that this population can be expanded to re-populate damaged tissue [37]. The cells isolated from the heart can be induced to form cardiac myocytes, SMCs and endothelial cells with appropriate markers for each, which form new myocardium with functional improvement. These cells derive from different lineages so it is unclear how adult cells might give rise to each lineage, or why cells lying dormant do not mobilise in response to injury. Although the mechanisms that allow regeneration are not yet fully understood, treatment through the induction of regeneration may prove feasible.
Conclusions Atherosclerosis is a chronic inflammatory disorder driven by risk factors that cause oxidative and inflammatory mechanisms. The balance between atherosclerotic plaque stability, with enhanced vascular SMC proliferation and matrix formation, and plaque instability produced by excessive inflammation, cellular apoptosis and secretion of MMPs determines plaque fate and the risk of clinical events. There has been significant improvement in the treatment of atherosclerosis through the reduction of circulating cholesterol levels. Still further progress needs to be achieved. The metabolic syndrome of insulin resistance driven by an escalating pandemic of obesity is an emerging target for treatment. Oxidative mechanisms and inflammation also stand out as targets for treatment. General inflammation is unlikely to be a good target, except in specific situations such as cardiac transplant recipients, who develop proliferative vasculopathy, as it is likely to lead to decreased host immunity and increased susceptibility to infection. Coronary atherosclerosis is the major cause of HF. The role of abnormal cardiac Ca2þ handling in the pathogenesis of HF and CM is increasingly understood. Further insights are being gained into the origins of cardiac hypertrophy and the role of adrenergic receptor signalling, mechanical stress and apoptosis in HF. Although it is not normally possible to repair the human heart through cardiac cell division, recent progress suggests that stem cell technology may offer a potential therapy.
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14. Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, Bravo M, Mitchell DJ, Sobel RA, Steinman L et al.: The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 2002, 420:78-84. The study shows a mechanism for the anti-inflammatory activity of the statin class of drugs on T-lymphocytes. The statin class of drugs reduces the activity of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. These drugs are highly effective in reducing blood cholesterol levels and reducing the risk of atherosclerosis. Statins also suppress antigen specific T cell activation by the suppression of the secretion of Th1Th2 cytokines in the situation of a mouse model of multiple sclerosis. Evidence is presented that these effects operate through the cholesterol biosynthesis pathway as they are reversed by administration of mevalonate. Mevalonate is necessary for the synthesis of cholesterol, and for the isoprenylation of proteins. 15. Eisen HJ, Tuzcu EM, Dorent R, Kobashigawa J, Mancini D, Valantine-von Kaeppler HA, Starling RC, Sorensen K, Hummel M, Lind JM et al.: Everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients. N Engl J Med 2003, 349:847-858. The major intra-cellular sirolimus receptor is a 12kb, FK506-binding protein (FKBP12). It produces a gain-of-function effect by targeting rapamycin/sirolimus (mTOR), a lipid kinase. This causes dephosphorylation and inactivation of P70S-kinase, which, when activated, stimulates ribosomal protein synthesis. Sirolimus also prevents cell-cycle progression through upregulation of cyclin-dependent kinase inhibitor p27Kip1 leading to the arrest of cell growth. Their activities effectively block IL-2-stimulated lymphocyte division, and is the basis for the clinical use of sirolimus in the prevention of allograft rejection and treatment of allograft vasculopathy. 16. Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS et al.: Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 2003, 349:1315-1323. 17. Castrillo A, Joseph SB, Vaidya SA, Haberland M, Fogelman AM, Cheng G, Tontonoz P: Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism. Mol Cell 2003, 12:805-816. This study provides the first real mechanistic link between infection and atherosclerosis. Model organisms are shown to activate TLRs and enhance so-called reverse cholesterol transport by the LXR pathway that activates a plasma membrane transporter ABC1. This pumps cholesterol out of the cell for loading of HDL. 18. Buffon A, Biasucci LM, Liuzzo G, D’Onofrio G, Crea F, Maseri A: Widespread coronary inflammation in unstable angina. N Engl J Med 2002, 347:5-12. 19. Mosca L: C-reactive protein–to screen or not to screen? N Engl J Med 2002, 347:1615-1617. 20. Aviles RJ, Askari AT, Lindahl B, Wallentin L, Jia G, Ohman EM, Mahaffey KW, Newby LK, Califf RM, Simoons ML et al.: Troponin T levels in patients with acute coronary syndromes, with or without renal dysfunction. N Engl J Med 2002, 346:2047-2052. 21. Heeschen C, Dimmeler S, Hamm CW, van den Brand MJ, Boersma E, Zeiher AM, Simoons ML: Soluble CD40 ligand in acute coronary syndromes. N Engl J Med 2003, 348:1104-1111. 22. Freedman JE: CD40 ligand–assessing risk instead of damage? N Engl J Med 2003, 348:1163-1165. 23. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, Kannel WB, Vasan RS: Obesity and the risk of heart failure. N Engl J Med 2002, 347:305-313. 24. Schmitt JP, Kamisago M, Asahi M, Li GH, Ahmad F, Mende U, Kranias EG, MacLennan DH, Seidman JG, Seidman CE: Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science 2003, 299:1410-1413. This study provides a direct link in the regulation of the cardiac sarcomeric 2þ Ca pump and the origins of DCM and HF. 25. Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME et al.: Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 2003, 421:634-639. Current Opinion in Genetics & Development 2004, 14:271–279
26. Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N et al.: Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med 2003, 9:1477-1483. Mice that develop DCM after deletion of the cell death protein PD-1 develop autoantibodies against cardiac TnI, a key element of the sarcomeric myofilaments, and also a target of inherited CM. 27. Eriksson U, Ricci R, Hunziker L, Kurrer MO, Oudit GY, Watts TH, Sonderegger I, Bachmaier K, Kopf M, Penninger JM: Dendritic cell-induced autoimmune heart failure requires cooperation between adaptive and innate immunity. Nat Med 2003, 9:1484-1490. 28. Hoshijima M, Ikeda Y, Iwanaga Y, Minamisawa S, Date MO, Gu Y, Iwatate M, Li M, Wang L, Wilson JM et al.: Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med 2002, 8:864-871. The authors show that pseudophosphorylated PLN transduction on a viral vector significantly suppresses HF in the mouse model for DCM. Pseudophosphorylated PLN is constitutively active. This effectively detaches PLN from the SERCA2a pump allowing calcium to move into the SR more quickly and causing the heart to beat more efficiently. The gene encoding this mutated protein is transduced into the heart. It markedly improves model heart failure phenotypes. 29. Lowes BD, Gilbert EM, Abraham WT, Minobe WA, Larrabee P, Ferguson D, Wolfel EE, Lindenfeld J, Tsvetkova T, Robertson AD et al.: Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents. N Engl J Med 2002, 346:1357-1365. RNAs encoding proteins that mediate Ca2þ uptake by the SR are favourably altered in patients with idiopathic DCM that show beneficial response to treatment. Myocardial gene expression was evaluated in subjects with idiopathic DCM after treatment with beta blockers. Those patients who benefited from this treatment showed increases in mRNAs encoding the SERCA2a routine and a-myocin heavy chain and a decrease in b-myocin heavy chain mRNA. These alterations in gene expression are consistent with increased activity of the encoded proteins leading to beneficial cardiac contraction. 30. Small KM, Wagoner LE, Levin AM, Kardia SL, Liggett SB: Synergistic polymorphisms of beta1- and alpha2Cadrenergic receptors and the risk of congestive heart failure. N Engl J Med 2002, 347:1135-1142. Patients who are double homozygous for mutations of adrenergic receptors are at substantially increased risk of HF. Black subjects, homozygous for common variants b1 and a2c adrenergic receptor genes, show marked increased in susceptibility to HF (gain of function). 31. Mialet Perez J, Rathz DA, Petrashevskaya NN, Hahn HS, Wagoner LE, Schwartz A, Dorn GW, Liggett SB: Beta 1-adrenergic receptor polymorphisms confer differential function and predisposition to heart failure. Nat Med 2003, 9:1300-1305. 32. Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H et al.: Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 2002, 8:35-40. A mechanism for cardiac hypertrophy induced by adrenergenic receptor signalling is provided. ADAM12 is a disintegrin metaloprotein that leads to the release of HB-EGF on signalling through a GPCR acting through the activation of protein kinase C. The HB-EGF is then available to transactivate the EGF receptor (ErbB1). 33. Brancaccio M, Fratta L, Notte A, Hirsch E, Poulet R, Guazzone S, De Acetis M, Vecchione C, Marino G, Altruda F et al.: Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat Med 2003, 9:68-75. 34. Yussman MG, Toyokawa T, Odley A, Lynch RA, Wu G, Colbert MC, Aronow BJ, Lorenz JN, Dorn GW II: Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy. Nat Med 2002, 8:725-730. 35. Leinwand LA: Hope for a broken heart? Cell 2003, 114:658-659. 36. Poss KD, Wilson LG, Keating MT: Heart regeneration in zebrafish. Science 2002, 298:2188-2190. www.sciencedirect.com
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37. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K et al.: Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003, 114:763-776. 38. Mamdouh Z, Chen X, Pierini LM, Maxfield FR, Muller WA: Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis. Nature 2003, 421:748-753. 39. Boucher P, Gotthardt M, Li WP, Anderson RG, Herz J: LRP: role in vascular wall integrity and protection from atherosclerosis. Science 2003, 300:329-332. 40. Glass CK, Witztum JL: Atherosclerosis: the road ahead. Cell 2001, 104:503-516. 41. Nabel EG: Cardiovascular disease. N Engl J Med 2003, 349:60-72. 42. Dwyer JH, Allayee H, Dwyer KM, Fan J, Wu H, Mar R, Lusis AJ, Mehrabian M: Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis. N Engl J Med 2004, 350:29-37. Leukotrienes are pro-inflammatory agents that recruit leukocytes. Variation in the gene that encodes a key regulatory enzyme of leukotriene biosynthesis is shown to be pro-atherogenic. This activity is linked to worsening disease in a diet rich in arachidonic acid. 43. Wickelgren I: Heart disease. Gene suggests asthma drugs may ease cardiovascular inflammation. Science 2004, 303:941. 44. Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S, Jonsdottir H, Thorsteinsdottir U, Samani NJ, Gudmundsson G, Grant SF, Thorgeirsson G et al.: The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat Genet 2004, 36:233-239. Variation of a gene responsible for the activation of the enzyme responsible for leukotriene biosynthesis is also associated with increased atherosclerosis. See also [42]. The gene encoding 5-lipoxygenase activating protein is associated with genetic variation conferring increased risk of myocardial infarction and stroke. This protein designated FLAP acts together with 5-lipoxygenase in the early stages of leukotriene biosynthesis.
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45. Gretarsdottir S, Thorleifsson G, Reynisdottir ST, Manolescu A, Jonsdottir S, Jonsdottir T, Gudmundsdottir T, Bjarnadottir SM, Einarsson OB, Gudjonsdottir HM et al.: The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nat Genet 2003, 35:131-138. 46. Ozaki K, Ohnishi Y, Iida A, Sekine A, Yamada R, Tsunoda T, Sato H, Hori M, Nakamura Y, Tanaka T: Functional SNPs in the lymphotoxin-alpha gene that are associated with susceptibility to myocardial infarction. Nat Genet 2002, 32:650-654. 47. Yamada Y, Izawa H, Ichihara S, Takatsu F, Ishihara H, Hirayama H, Sone T, Tanaka M, Yokota M: Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002, 347:1916-1923. 48. Wang L, Fan C, Topol SE, Topol EJ, Wang Q: Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science 2003, 302:1578-1581. 49. Broeckel U, Hengstenberg C, Mayer B, Holmer S, Martin LJ, Comuzzie AG, Blangero J, Nurnberg P, Reis A, Riegger GA et al.: A comprehensive linkage analysis for myocardial infarction and its related risk factors. Nat Genet 2002, 30:210-214. 50. Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, Willeit J, Schwartz DA: Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002, 347:185-192. Variation of the TLR4 reduces the risk of atherosclerosis but increases the risk of infection. This is the first description that links genetic variation in susceptibility to infection by gram negative organisms to the risk of atherosclerosis. The TLRs are key receptors for gram negative bacterial lipopolylsaccharides. The variation of these receptors that reduces their efficacy in binding these bacterial products markedly reduces host defence, particularly in early life. However, subjects with these common variants later in life develop less atherosclerosis. This provides the first very important major genetic link between infection and inflammation and atherogenesis. 51. Herrington DM, Howard TD, Hawkins GA, Reboussin DM, Xu J, Zheng SL, Brosnihan KB, Meyers DA, Bleecker ER: Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Engl J Med 2002, 346:967-974.
Current Opinion in Genetics & Development 2004, 14:271–279
Introduction Cardiovascular disease, including heart attack, stroke and heart failure (HF), is the leading cause of disease and death in the developed world, and is poised to become the most significant health problem worldwide. Atherosclerosis is the major cause of cardiovascular disease. Table 1 shows the risk factors for atherosclerosis and the main patho-biological processes on which they converge. This review concentrates on the pathogenesis of atherosclerosis as a disease driven by oxidative stress and enhanced inflammation in the artery wall. The role of inflammation in lesion progression and in current and emerging treatment measures is described. New markers of disease activity and myocardial infarction are described. Atherosclerosis is also the driver of the emerging epidemic of HF. The role of abnormal calcium handling in this disorder is described along with factors that lead to cardiac hypertrophy and HF as well as the potential of stem cell therapy for repairing the damaged heart.
Pathogenesis of atherosclerosis
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Autopsy studies show that atherosclerosis develops slowly over many years. A similar disease is produced in experimental animals, where a high-cholesterol diet and genetic manipulation cause accelerated lesions. These studies have produced a picture of the evolution of atherosclerosis from fatty streak to the complex plaque and clinical disease (Figure 1).
This review comes from a themed issue on Genetics of disease
Oxidative processes
Addresses Imperial College London, South Kensington, London SW7 2AZ, UK e-mail: [email protected]
0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.04.012
Abbreviations CM cardiomyopathy CRP C-reactive protein DCM dilated cardiomyopathy ER endoplasmic reticulum GPCR G-protein-coupled receptor HB-EGF heparin-binding epidermal growth factor HCM hypertrophic cardiomyopathy HDL high-density lipoprotein HF heart failure LDL low-density lipoprotein MMPs matrix metalloproteases PLN phospholamban PPAR peroxisome proliferator activated receptor SERCA2a SR Ca2þ-ATP SMC smooth muscle cell SR sarcoplasmic reticulum TLRs Toll-like receptors www.sciencedirect.com
LDL and other nutrient macromolecules are transported in caveolae through endothelial cells into the vascular intima. LDL is retained in the sub-endothelium by a charged interaction with matrix proteoglycans [1]. Trapped LDL is attacked and oxidised by reactive oxygen species (superoxide anion, hydrogen peroxide, lipid peroxides and peroxynitrite), of which ozone is the latest and most malignant member of the ‘family’ [2]. Reactive oxygen species are inactivated by dietary antioxidants and enzymes such as glutathionine peroxidase, the principal cellular anti-oxidant enzyme [3]. Low levels of this enzyme predispose to atherosclerosis. High oxidative stress is also found in hypertension (mediated through abnormal renal salt handling and increased production of angiotensin II) and cigarette smoking (as a result of reduced glutathione peroxidase) [3,4]. This provides a compelling convergence of mechanisms for the major risk factors for atherosclerosis (Table 1). Inflammation and lesion formation
Oxidized LDL is engorged by macrophages, which take on a characteristic foamy appearance, and become Current Opinion in Genetics & Development 2004, 14:271–279
272 Genetics of disease
Table 1 Risk factors for atherosclerosis. Risk factors
Primary pathogenetic process
Not modifiable Age Ethnicity Male gender Genetics Modifiable by lifestyle change Diet high in saturated fat and cholesterol, low in fruit, vegetables and grain Obesity Smoking Lack of exercise Modifiable by drugs Dyslipidaemia Hypertension Non-traditional Lipoprotein (a) Homocysteine Infection Systemic lupus erytyematosis
recruited and induced to proliferate and secrete collagen by growth factors such as PDGF and TGF-b. In time, the characteristic fatty-fibrous lesion of atherosclerosis develops (Figure 1). Lesion progression
High blood cholesterol, oxidative stress Metabolic syndrome of insulin resistance Oxidative stress Poor perfusion, adverse lipid profile Oxidative stress Oxidative stress, enhanced vasoconstriction Thrombogenesis Oxidative stress Inflammation Inflammation
activated leading to a state of chronic inflammation (Figure 1) [5]. Cytokines (IL-1, IFN-g, TNF-b, and angiotensin II) and chemokines (MCP-1, IL-8, IFN-g, CXC cytokines, and eotaxin) promote adhesion molecule expression (VCAM-1, P- and E-selectin) and attract immune and inflammatory cells (monocytes, leukocytes, B and T lymphocytes, and mast cells), mainly in regions of turbulent blood flow. Leukocyte attachment to adhesion molecules causes them to move out of the blood stream and roll along the surface of endothelial cells. They egress from the blood at endothelial cell tight junctions (Figure 1). Smooth muscle cells (SMCs) are
The growth of the atheromatous plaque is not a continuous process. Lesions undergo extensive remodelling, and neo-vascularisation, punctuated by bursts of activity and regression, which may produce cardiac events. The stable plaque has a preponderance of SMCs embedded in a dense matrix of collagen. Such plaques are typically achieved by lipid-lowering therapy. They are low in inflammatory cell content and cytokines such as bFGF, VEGF and PDGF promote endothelial cell and SMC survival [6]. The unstable plaque usually has a large core of inflammatory cells and debris from dead cells. The cap of these plaques is often fragile and denuded of endothelial cells. These lesions are subject to excessive inflammation. Inflammatory cells produce pro-apoptotic cytokines, such as TNF-b, for SMC and endothelial cells, matrix metalloproteases (MMPs), which digest collagen fibrils, and cytokines, such as IFN-g, which inhibit collagen secretion from SMCs. Cholesterol accumulation in macrophages signals death by perturbing the ER membranes, which depletes Ca2þ stores and induces an unfolding protein response, leading to activation of the cell death effector CHOP [7]. These characteristics make such plaques particularly prone to physical disruption. Although plaque rupture is the cause of clinical events, in many cases it is silent (i.e. cause no clinical symptoms). In such silent events, the thrombus generated is absorbed into the existing plaque. Plaque growth is then stimulated by the release from platelets of PDGF and TGF-b, which together stimulate SMC recruitment and proliferation and the production of a dense collagen matrix. Remodelling is also mediated through autocrine effects of PDGF and
(Figure 1 Legend) Progressive cellular and molecular mechanisms in the origins of atherosclerotic plaque, and common processes involved in hypertrophy, dilatation and heart failure. (a) The three diagrams show the development and progression of atherosclerotic lesions from fatty streak, to fibrous plaque and ruptured plaque. LDL is trapped in the sub-endothelial space, where it undergoes oxidation. Monocytes attach to endothelial cells and migrate into the intima through tight junctions. This process involves an intimate interaction with the junctional adhesion molecule PECAM and the release of spare membrane from cellular invaginations [38]. Monocytes become activated, and express the scavenger receptors CD36 and SR-A, which promote the uptake of oxidised LDL. Homeostatic responses facilitate reverse cholesterol transport through HDL. Macrophages signal the recruitment and activation of T-lymphocytes, the two cells together perform the concert of activity that leads to atherosclerosis. This involves SMC, controlled by interaction between PDGFR (platelet derived growth factor receptor) and LRP (low density lipoprotein related protein [39], recruitment and proliferation and production of the dense plaque matrix [39]. Pro-inflammatory and pro-apoptotic processes and physical disruption pre-dispose to plaque rupture. Macrophage secretion of MMPs and microvascularisation contribute to plaque weakness and susceptibility to rupture. Plaque rupture exposes blood components to tissue factor initiating coagulation and the recruitment of platelets. Formation of thrombus may lead to catastrophic clinical events or resolution by incorporation into plaque. After Glass and Witztum [40]. (b) The principal causes of cardiac hypertrophy are listed. Hypertrophic cardiomyopathy (HCM) is the common cause of sudden death in adolescents and young adults, especially in athletes [41]. It is an autosomal dominant disorder affecting sarcomeric proteins, which leads to disruption of force transmission and cellular hyperplasia, and hypertrophy of the ventricles. Cardiac function deteriorates as myocytes undergo apoptosis, followed by scar formation leading to sudden death or heart failure. The principal causes of cardiac dilatation are listed. Dilated cardiomyopathy (DCM) is the most frequent form of cardiomyopathy. It occurs mainly in children. DCM has both acquired and genetic causes. Adenovirus or coxsacchie virus infection can progress to autoimmune destruction of the myocardium and expansion of the ventricles. Inherited defects of a variety of proteins can cause similar changes in the ventricles. These include several sarcomeric proteins, also associated with HCM [41]. Current Opinion in Genetics & Development 2004, 14:271–279
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Pathophysiology and biochemistry of cardiovascular disease Scott 273
Figure 1
(b)
(a) Vascular lumen
Adhesion VCAM-1 P-selectin E-selectin ICAM-1 PECAM
Monocyte
Endothelial cells
MCP-1 CCR-2 IL-8 CXC
Extracellular matrix
Intima
Lymphocytes Migration LDL HDL LDL oxidation
Differentiation M-CSF
mmLDL
15 LO INOS Ozone
MMPs
Homeostatic responses
Tissue factor
apo E ABC A1 ACAT
Cytokines
Foam cell
ROS
ox LDL
Cardiac hypertrophy
Internal elastic lamina ox LDL uptake
Hypertension Hypertrophic cardiomyopathy
scaveger receptors CD36 SR-A
Media
Macrophage
Smooth muscle cells
Vascular lumen Endothelial cells Fibrinogen
Fibrous cap Fibrin Collagen Proteoglycans
Cytokines PDGF TGF-β
IL-4 IL-10
Macrophage foam cells
IL-1 IFN-γ Th2
PDGFR LRP
TNF-β CD40
IL-4 IL-10 Th1
Smooth muscle cells
Coronary arteries
Vascular lumen
Cardiac dilatation and heart failure
Platelets SMC-derived foam cells CD40 PDGF
Necrotic core CD40
Tissue factor
Atherosclerosis Hypertension Metabolic syndrome Dilated cardiomyopathy
Extracellular lipid 'Gruel'
Macrophage foam cell
MMPs
IFN-γ Th1 cell Current Opinion in Genetics & Development
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274 Genetics of disease
New micro-vessels in the plaque are fragile and likely to rupture, generating intra-plaque haemorrhage, which may resolve contributing further to plaque growth, or accrete cholesterol from red cell membranes, which contribute to plaque instability [10].
which is suppressed by PPARa and g agonists. PPARd, another member of the family, enhances fat burning in fatty tissue and muscle [11,12]. It also represses macrophage activity by a novel mechanism in which the binding of ligand to the nuclear receptor PPARd leads to release of BCL-6, a transcriptional repressor. In the absence of ligand, BCL-6 remains bound to PPARd resulting in a pro-inflammatory response.
Management of atherosclerosis
Oxidation and inflammation
Established treatment
For many non-traditional risk factors, the strength of evidence supporting their role in atherogenesis is not strong, perhaps because their role is either modest or complex (Table 1). For example, oxidative stress is a pervasive unifying mechanism in atherogenesis, but clinical trials using anti-oxidants show uniformly negative results. However, a recent large prospective population study showing the benefits of traditional Mediterranean diet, and its presumed suppression of oxidative stress, suggests that the situation is more complex, and that a varied diet provides the necessary anti-oxidants, which simple supplementation does not [13].
TGF-b, which operate under the control of the retinoicacid receptor and Kru¨ ppel-like transcription factors [8,9].
Reduction of elevated levels of circulating LDL cholesterol is the best treatment we have for atherosclerosis. However, LDL lowering decreases the risk of cardiac events by only 30%, even with optimum treatment of other major risk factors. This is perhaps because disease is well established by the time treatment is started. Moreover, target ‘normal’ cholesterol levels are probably high for our species, as compared to human neonates and animals in the wild. Elucidation of atherogenic mechanisms, particularly those involved in oxidation and inflammation, and better evaluation of non-traditional risk factors such as lipoprotein (a) and infection, may in the future lead to improved therapy (Table 1). Some recent insights from genetics are shown in Table 2. Emerging risk factors
A prominent and serious risk factor is the metabolic syndrome of insulin resistance (Table 1), which is driven by obesity. Insulin resistance leads to diabetes, with the generation of highly atherogenic advanced glycation end products, an aggressive atherogenic lipoprotein profile (hypertriglyceridaemia, reduced HDL [high-density lipoprotein], and readily oxidised, small dense LDL), hypertension, and a pro-coagulant state. Adipocytes in the obese have many parallels with macrophage foam cells. In the obese, macrophages also pervade fatty tissue. Both cell types engage in lipid storage and cytokine secretion,
Inflammation is now seen as so central to atherogenesis that it is an obvious therapeutic target [5]. Indeed, the drugs used for the treatment of atherosclerosis — aspirin, statins, PPARa agonists and angiotensin II signalling inhibitors — all have anti-inflammatory effects [5,14]. However, the use of broad-spectrum anti-inflammatory or immunosuppressive drugs for the treatment of a chronic disease like atherosclerosis compromises host defences. The successful treatment of cardiac transplant vasculopathy illustrates this. Transplant vasculopathy is a relentlessly progressive diffuse SMC proliferation. It is treated successfully with a new class of macrocyclic immunosuppressive agents with unique anti-proliferative activity, but this success is compromised by a high rate of bacterial infection (see also Table 2: TLR4, genetic variation,
Table 2 Genetic studies and atherosclerosis. Gene defect
Clinical syndrome, molecular mechanism
Refs
(a)
Atherosclerosis, increased leukotrienes, increased inflammation recruitment of T-cells and macrophages, increased vascular permeability enhanced by dietary arachidonic acid Common stroke, atherosclerosis, degradation cAMP, immune modulation Myocardial infarction, cytokine induction of adhesion molecules Myocardial infarction, gap junction between endothelial cells Myocardial infarction, fibrinolysis Myocardial infarction, MMP Myocardial infarction, proliferation of SMCs, co-operates with GATA1 to control the expression of atrial natriuretic factor Myocardial infarction gene not identified Atherosclerosis decreased, innate immune response to gram negative organism decreased, susceptibility to infection increased Augmented response of HDL cholesterol to hormone replacement therapy
[42] [43,44] [45] [46] [47] [47] [47] [48]
5-lipoxygenase activation 5-lipoxygenase activating protein (b) Phosphodiesterase 4D (c) Lymphotoxin-a (c) Connexin 37 (c) Plasminogen activator inhibitor 1 (c) Stromelysin (b) Myocyte enhancer factor 2 (b)
(b) (a)
Linkage chromosome 14 Toll-like receptor 4
(a)
Estrogen Receptor
(a)
(b)
Candidate gene;
linkage analysis;
(c)
[49] [50] [51]
large-scale association study.
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Pathophysiology and biochemistry of cardiovascular disease Scott 275
infection and atherosclerosis) [15]. Nonetheless, the macrocyclic agents are highly successful for short-term, local use in the prevention of endothelial cell and SMC proliferation after stenting at the time of coronary angioplasty [16]. In consideration of possible new therapeutic targets, the redundancy of distal effectors of inflammation suggest that narrow-spectrum distal inhibition will not modify disease effectively. Rather, the targeting of proximal triggers, such as ozonolysis [2], may prove a more promising strategy for interrupting inflammation in atherogenesis [5]. Infection
A variety of studies link acute respiratory infections and organisms such as Chlamydia pneumonae, Helicobacter pylori and cytomeglalovirus to atherosclerosis, but the role of pathogens remains unclear. Recent studies make an important link [17]. Bacterial lipopolylsaccharides are cleared by Toll-like receptors (TLRs), which are crucial for defence against infection. TLR stimulation releases antimicrobial peptides, cytokines, and initiates adaptive immunity. TLRs also block the activation of the nuclear receptor LXR (liver X nuclear receptor). This decreases the activity of the ABC1 transporter and pre-disposes macrophages to foam cell formation (see also Table 2: genetic variation, infection and atherosclerosis). Infection is also considered as a link to the general vascular inflammation that accompanies unstable angina [18]. Markers of atherosclerosis and myocardial infarction
C-reactive protein (CRP) is an important marker of general inflammation and of atherosclerosis [19]. It is independent of other markers such as LDL. CRP is produced by the liver in response to cytokines such as IL-6. It binds LDL/ oxidised LDL and is present in lesions, where it may act as a chemoattractant, and be involved in the expression of adhesion molecules. Its role as a marker for predictive screening of atherosclerosis, which contributes to other markers, remains to be established in randomised clinical trials. Whether markers such as products of ozonolysis will predict atherosclerosis requires clarification [2]. The bulk of cardiac troponin T forms an important component of the myocardial sarcomere (see Calcium handling in disease, below); the remainder is free in the cytoplasm [20]. When myocardial damage occurs, the cytoplasmic pool is released, followed by the protracted release of myofilament tropinin. Troponin is a much more sensitive measure of cardiac damage than the conventional MB isoenzyme fraction of myocardial creatine kinase. Cardiac troponin levels in the blood predict short-term prognosis in acute coronary syndromes particularly in microinfarction. Soluble CD40 ligand, a member of the TNF family, is another predictor of acute coronary syndromes [21,22]. Soluble CD40 ligand is expressed on leukocytes, www.sciencedirect.com
endothelial cells, smooth muscle cells and activated platelets. Although not a general marker of atherosclerosis, it appears to predict acute coronary syndromes and is independent of troponin. Elevation of soluble CD40 ligand indicates platelet activation and increased risk of cardiovascular events. Patients with elevated CD40 levels should benefit from anti-platelet treatment.
The heart HF has for the patient a prognosis worse than many forms of cancer [23,24]. Coronary atherosclerosis, hypertension and the metabolic syndrome are the major causes of HF. The primary cardiomyopathies (CMs) and arrhythmias are rare, but have received considerable attention because of their often dramatic presentation with sudden death, increased frequency in children, and the important insights they provide into common HF (Figure 1). Normal calcium handling
A frequent common feature of HF, CM and cardiac arrhythmia is abnormal myocyte Ca2þ signalling. Normal cardiac contractions are initiated by the entry of Ca2þ into cells. This leads to the release of large amounts of Ca2þ required for muscle contraction from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR/RyR2). A member of the immunophilin family of cytosolic binding proteins, FK506 binding protein (FKBP12/12.6), stabilises RYR preventing aberrant activation of the channel during the resting phase of the cardiac cycles. During exercise, RYR phosphorylation by protein kinase A dissociates FKPB12/12.6 from the channel increasing Ca2þ release and cardiac contractility. Variation in Ca2þ concentration is transferred to the thin sacromeric filaments (actin, tropomycin and troponin) by the Ca2þ receptor troponin. Three genes encode the troponin subunits for Ca2þ binding (TnC), inhibition (TnI), and tropomysin-binding (TnT). The structure of troponin in the Ca2þ-saturated form provides important insights into the role of troponin in the initiation of contraction. At low intracellular Ca2þ concentration, TnI interacts with actin and blocks the actomyosin ATPase. Increased Ca2þ binding to TnC releases TnI from its interaction with actin so that inhibition is released and contraction initiated. Cardiac contraction ends when Ca2þ is pumped back into the SR. This is regulated by the protein phospholamban (PLN), which in its unphosphorylated form sits on and inhibits the SR Ca2þ-ATP (SERCA2a) pump. Phosphorylation of PLN, in response to signals such as adrenaline, detaches PLN from the pump, allowing Ca2þ to move into the SR more quickly, and causing the heart to beat more efficiently. Calcium handling in disease
Mutations in the genes encoding Naþ and Kþ channels involved in cardiac action potentials underlie fatal cardiac arrhythmias in inherited long QT syndrome. Defects in proteins involved in Ca2þ transport also cause fatal arrhythmia. Mutation of ankryn B causes long QT Current Opinion in Genetics & Development 2004, 14:271–279
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syndrome and arrhythmia in humans and mice, by disruption of the cellular organisation of the Naþ pump, the Naþ/Ca2þ exchanger and the inositol-1, 4, 5-tryphosphate receptors (all ankryn B binding proteins) [25]. Mutations in the RyR2 Ca2þ channel also trigger fatal cardiac arrhythmias. Recent studies help clarify the link between acute infection and chronic autoimmune injury, and abnormal Ca2þ handling in both primary and acquired dilated cardiomyopathy (DCM). The autoimmune induction of antibodies against cardiac TnI, which is also defective in primary DCM, and the administration of monoclonal antibodies to TnI induce DCM in wild-type mice [26,27]. Antibodies against TnI augment the voltage-dependent L-type calcium current of cardiomyocytes, suggesting that antibodies to TnI induce DCM by chronic stimulation of calcium influx in cardiomyocytes. Mutation of the PLN gene leads to DCM with refractory HF. Cellular and biochemical studies indicate the mutant PLN, unlike wild-type, does not inhibit SERCA2a directly (Figure 1) [24]. This suggests that therapies aimed at preventing PLN from inhibiting the SERCA2a pump might be used to treat ordinary HF. Indeed HF progression in DCM is suppressed by a pseudo-phosphorylated mutant of a PLN via in vivo cardiac rAAV gene delivery [28]. In line with these findings in mice, patients with idiopathic DCM, who improve on b blocker treatment, show increases in SERCA2a and a-myocin heavy chain mRNAs and a decrease in b-myocin heavy chain mRNA, changes consistent with our understanding of the pathogenesis of disease, and the role of Ca2þ handling in this [29]. Cardiac hypertrophy and heart failure
Pressure overload in hypertension is triggered by two types of input that result in cardiac hypertrophy: neurohumoral mediators that activate growth factor receptor tyrosine kinases, cytokine receptors and G-proteincoupled receptors (GPCRs); and biomechanical stress. Stimulation of adrenergic receptors results in activation of G proteins, which can signal by triggering the synthesis of cAMP. This leads to the activation of Ca2þ regulatory proteins (including the plasma membrane Ca2þ channel and PLN) and myofilament proteins. This, in turn, leads to altered sarcomeric protein synthesis and cardiac hypertrophy, which may be beneficial as it allows the heart to work harder. However, it may also reduce the efficiency of structural adaptation leading to dilatation and HF. Double homozygotes for gain of function genetic variation of the b1 and a2C-adrenergic receptors produces substantial increased risk of HF in patients [30,31]. To clarify the mechanism of these processes, a mouse model of the b1 receptor variant has been generated. Importantly the mice, as do humans with this SNP, show Current Opinion in Genetics & Development 2004, 14:271–279
enhanced b-blocker responsiveness and reduced cardiac deterioration, thus indicating a preventative treatment strategy. GPCR signalling is now linked to cardiac hypertrophy. GPCR signalling can also operate through phospholipase C, which generates diacylglycerol. Diacylglycerol is a potent activator of protein kinase C, whose association with a distintergin metalloprotein called ADAM12 leads to the shedding of membrane-bound heparin-binding epidermal growth factor (HB-EGF) [32]. HB-EGF is then available to transactivate the EGF receptor, ErbB1, which stimulates cellular growth leading to cardiac hypertrophy. By contrast, the antibody herceptin (used in the treatment of breast cancer) that blocks the ErbB2 receptor gives rise to CM. The generation of mice with ventricle-restricted deletion of ErbB2 also show CM. Together these results indicate that ErbB1 and -2 signalling determine the balance between cardiac hypertrophy and cardiac dilatation. The role of biomechanical sensors in cardiac hypertrophy is less well understood than neuro-hormonal mechanisms. Transduction of mechanical stress into biochemical signals is mediated largely by integrins that couple the extracellular matrix to the cellular cytoskeleton. An important protein that acts as a biomechanical sensor in this pathway is melusin, which interacts with the cytoplasmic domain of the integrins, and is expressed only in skeletal and cardiac muscle [33]. Mice deficient for melusin still show cardiac hypertrophy in response to hormones acting through GPCRs, but not in response to biomechanical stress. However, in response to biomechanical pressure overload, melusin-deficient mice develop DCM and HF. These results indicate that melusin protects against HF, probably activating protective pathways that allow cardiac hypertrophy. The loss of cardiomyocytes through apoptosis is a key event in the development of HF. The molecular mechanisms that induce cell death have now been elucidated in pressure overload cardiac hypertrophy by gene-expression profiling. The mitochondrial death protein Nix is induced and triggers apoptotic cardiomyopathy in Gqmediated pressure overload cardiac hypertrophy [34]. These data establish a molecular mechanism for induction of cardiomyocyte apoptosis in hypertrophied myocardium and for its progression to HF. Repair of the damaged heart
Amphibians and zebrafish can regenerate heart tissue but mammals cannot [35,36]. Thus, after myocardial infarction, and in CM and HF there is permanent cell loss. A remedy for this would be to induce the heart to make new cells by the division of existing cells. One strain of mouse can apparently regenerate and a series of genes have been identified that allow this. Considerable debate exists as to www.sciencedirect.com
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whether circulating or resident stem cells can differentiate into mature cardiac tissue in humans. A recent study suggests a stem cell population exists in the adult heart and that this population can be expanded to re-populate damaged tissue [37]. The cells isolated from the heart can be induced to form cardiac myocytes, SMCs and endothelial cells with appropriate markers for each, which form new myocardium with functional improvement. These cells derive from different lineages so it is unclear how adult cells might give rise to each lineage, or why cells lying dormant do not mobilise in response to injury. Although the mechanisms that allow regeneration are not yet fully understood, treatment through the induction of regeneration may prove feasible.
Conclusions Atherosclerosis is a chronic inflammatory disorder driven by risk factors that cause oxidative and inflammatory mechanisms. The balance between atherosclerotic plaque stability, with enhanced vascular SMC proliferation and matrix formation, and plaque instability produced by excessive inflammation, cellular apoptosis and secretion of MMPs determines plaque fate and the risk of clinical events. There has been significant improvement in the treatment of atherosclerosis through the reduction of circulating cholesterol levels. Still further progress needs to be achieved. The metabolic syndrome of insulin resistance driven by an escalating pandemic of obesity is an emerging target for treatment. Oxidative mechanisms and inflammation also stand out as targets for treatment. General inflammation is unlikely to be a good target, except in specific situations such as cardiac transplant recipients, who develop proliferative vasculopathy, as it is likely to lead to decreased host immunity and increased susceptibility to infection. Coronary atherosclerosis is the major cause of HF. The role of abnormal cardiac Ca2þ handling in the pathogenesis of HF and CM is increasingly understood. Further insights are being gained into the origins of cardiac hypertrophy and the role of adrenergic receptor signalling, mechanical stress and apoptosis in HF. Although it is not normally possible to repair the human heart through cardiac cell division, recent progress suggests that stem cell technology may offer a potential therapy.
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