THE CONCEPT OF ATHEROSCLEROTIC RISK FACTORS
The systematic study of risk factors for atherosclerosis emerged from a coalescence of experimental results, as well as from cross-sectional and ultimately longitudinal studies in humans. The prospective, community-based Framingham Heart Study provided rigorous support for the concept that hypercholesterolemia, hypertension, and other factors correlate with cardiovascular risk. Similar observational studies performed worldwide bolstered the concept of “risk factors” for cardiovascular disease.
From a practical viewpoint, the cardiovascular risk factors that have emerged from such studies fall into two categories: those modifiable by lifestyle and/or pharmacotherapy, and those that are immutable, such as age and sex. The weight of evidence supporting various risk factors differs. For example, hypercholesterolemia and hypertension certainly predict coronary risk, but the magnitude of the contributions of other so-called nontraditional risk factors, such as levels of homocysteine, levels of lipoprotein (a) [Lp(a)], and infection, remains controversial. Moreover, some biomarkers that predict cardiovascular risk may not participate in the causal pathway for the disease or its complications. Genetic studies using genome-wide association (GWAS) approaches and Mendelian randomization approaches have helped to distinguish between risk markers and factors that contribute causally to the disease. For example, recent genetic studies suggest that C-reactive protein (CRP) does not itself mediate atherogenesis, despite its ability to predict risk, whereas Lp(a) and apolipoprotein C3 have emerged as a causal risk factor. Table 291e-1 lists a number of risk factors implicated in atherosclerosis. The sections below will consider some of these factors and approaches to their modification.
TABLE 291e-1Major Risk Factors for Atherosclerosis ||Download (.pdf) TABLE 291e-1Major Risk Factors for Atherosclerosis
|High LDL cholesterol |
|Cigarette smoking |
|Hypertension (BP ≥140/90 mmHg or on antihypertensive medication) |
|Low HDL cholesterola (<1.0 mmol/L [<40 mg/dL]) |
|Diabetes mellitus |
|Family history of premature CHD |
|Age (men ≥45 years; women ≥55 years) |
|Lifestyle risk factors |
| Obesity (BMI ≥30 kg/m2) |
| Physical inactivity |
| Atherogenic diet |
|Emerging risk factors |
| Lipoprotein (a) |
| Prothrombotic factors |
| Proinflammatory factors |
| Impaired fasting glucose |
| Subclinical atherosclerosis |
Abnormalities in plasma lipoproteins and derangements in lipid metabolism rank among the most firmly established and best understood risk factors for atherosclerosis. Chapter 421 describes the lipoprotein classes and provides a detailed discussion of lipoprotein metabolism.
The American College of Cardiology and American Heart Association (ACC/AHA) promulgated new guidelines on risk assessment, lifestyle measures, and cholesterol management in 2013. The panels that produced these guidelines followed an evidence-based approach. The 2013 cholesterol guideline focused on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) rather than other classes of lipid-modulating drugs, including fibric acid derivatives, cholesterol absorption inhibitors such as ezetimibe, and niacin products. The guideline cites the lack of contemporary randomized clinical trial evidence that supports the efficacy of these nonstatin lipid-modifying agents in cardiovascular event reduction. The cholesterol guideline defined four statin benefit groups (Table 291e-2): (1) all individuals who have clinical atherosclerotic cardiovascular disease (ASCVD), therefore considered “secondary prevention”; (2) those with LDL cholesterol ≥190 mg/dL without a secondary cause such as a high intake of saturated or trans fats, various drugs, or certain diseases; (3) individuals with diabetes without established cardiovascular disease who are 40–75 years old and have LDL cholesterol of 70–189 mg/dL; and (4) those without established ASCVD without diabetes who are 40–75 years old and who have LDL cholesterol of 70–189 mg/dL and a calculated ASCVD risk ≥7.5%. An online risk calculator based on pooled cohorts was provided to aid clinicians and patients in calculating their risk (http://my.americanheart.org/professional/StatementsGuidelines/PreventionGuidelines/Prevention-Guidelines_UCM_457698_SubHomePage.jsp). Other validated risk calculators that incorporate family history of CAD and a marker of inflammation (high-sensitivity CRP [hsCRP]) that apply to U.S. women and men exist (http://www.reynoldsriskscore.org). Downloadable applications for risk calculation on handheld devices are readily available.
TABLE 291e-2Summary of the Four Statin Benefit Groups Described in the 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults ||Download (.pdf) TABLE 291e-2Summary of the Four Statin Benefit Groups Described in the 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults
Clinical ASCVD “secondary prevention”
LDL-C ≥190 mg/dL without secondary cause (e.g., saturated/trans fats, drugs, certain diseases)
Primary prevention with diabetes mellitus: age 40–75 years, LDL-C 70–189 mg/dL
Primary prevention without diabetes mellitus: age 40–75 years, LDL-C 70–189 mg/dL, estimated ASCVD risk ≥7.5%
The 2013 guideline emphasized a patient-centered approach and recommended that clinicians and patients engage in a risk-benefit conversation before starting statin therapy and not rely solely on calculated risks or arbitrary category assignment. It further emphasizes that medications do not supplant a healthy lifestyle. The guideline also provides some practical suggestions regarding management of muscle symptoms attributed to statins, an issue of considerable concern to many patients and practitioners alike.
In a major departure from prior guidelines, the 2013 guideline eliminates LDL targets as goals of therapy. The panel did so because major clinical trials did not titrate therapy to a goal, but rather used fixed doses of statins. Instead, the new guideline suggests different intensities of statin therapy based on risk category (Fig. 291e-4).
Major recommendations for statin therapy for atherosclerotic cardiovascular disease (ASCVD) prevention. LDL-C, low-density lipoprotein cholesterol. (From NJ Stone et al: J Am Coll Cardiol, 2013, doi: 10.1016/j.jacc.2013.11.002.)
The 2013 guideline focus on statins reflects an extensive body of rigorous evidence that supports the effectiveness of this class of drugs in cardiovascular event reduction and an acceptable risk-benefit relationship (Fig. 291e-5). Moreover, because almost all statins are now available as generic statins medications, cost has become much less of an impediment to their use.
The Cholesterol Treatment Trialists Collaboration meta-analyzed 27 randomized clinical trials evaluating statin therapy. They found profound decreases in both major vascular events and vascular death (not shown) proportional to the magnitude of low-density lipoprotein (LDL) cholesterol reduction achieved with statin treatment. This diagram shows the results of this meta-analysis for vascular death. (From Lancet 380:581, 2012.)
The clinical use of effective pharmacologic strategies for lowering LDL has reduced cardiovascular events markedly, but a considerable burden of residual risk remains even in patients treated with high-intensity statins. Hence, current studies are evaluating other avenues to address the residual burden of cardiovascular disease that persists despite statin treatment. Inhibitors of genetic studies identified proprotein convertase subtilisin kexin-like 9 (PCSK9) as a regulator of LDL levels associated with cardiovascular outcomes. Interaction of the LDL receptor with PCSK9 hastens the receptor’s degradation, and hence yields higher circulating LDL concentrations. Genetic variants that lower PCSK9 activity appear to protect against cardiovascular events. Monoclonal antibodies that neutralize PCSK9 lower LDL levels even in statin-treated patients and are currently under investigation as novel therapeutics to lower cardiovascular risk.
LDL-lowering therapies do not appear to exert their beneficial effect on cardiovascular events by causing a marked “regression” of stenoses. Studies of lipid lowering monitored by angiography or by intravascular imaging modalities have shown at best a modest reduction in coronary artery stenoses over the duration of study, despite abundant evidence of event reduction. These results suggest that the beneficial mechanism of lipid lowering by statins does not require a substantial reduction in the fixed stenoses. Rather, the benefit may derive from “stabilization” of atherosclerotic lesions without substantially decreased stenosis. Such stabilization of atherosclerotic lesions and the attendant decrease in coronary events may result from the egress of lipids or from favorably influencing aspects of the biology of atherogenesis discussed above. In addition, as sizable lesions may protrude abluminally rather than into the lumen due to complementary enlargement, shrinkage of such plaques may not be apparent on angiograms. The consistent benefit of statins may depend not only on their salutary effects on the lipid profile, but also on direct modulation of plaque biology independent of lipid lowering.
As the prevalence of metabolic syndrome and diabetes increases, many patients present with low concentrations of HDL (HDL cholesterol <1.0 mmol/L [<40 mg/dL]). A baseline measurement of HDL cholesterol indubitably correlates with future cardiovascular risk. Yet, the utility of therapies that raise HDL cholesterol levels in blood as effective interventions to reduce cardiovascular vascular events has come into question. Blood HDL levels vary inversely with those of triglycerides, and the independent role of HDL versus triglycerides as a cardiovascular risk factor remains unsettled. The 2013 guideline does not advocate any specific therapy for raising HDL. Indeed, multiple recent trials failed to show that raising HDL cholesterol levels improves cardiovascular outcomes, and recent genetic studies cast doubt on low HDL as a causal risk factor for atherosclerotic events. Weight loss and physical activity can raise HDL, and these lifestyle measures merit universal adoption (Table 291e-3). Nicotinic acid, particularly in combination with statins, can robustly raise HDL, but clinical trial data do not support the effectiveness of nicotinic acid in cardiovascular risk reduction. Agonists of nuclear receptors provide another potential avenue for raising HDL levels. Yet patients treated with peroxisome proliferator–activated receptors alpha and gamma (PPAR-α and -γ) agonists have not consistently shown improved cardiovascular outcomes, and at least some PPAR agonists have been associated with worsened cardiovascular outcomes. Other agents in clinical development raise HDL levels by inhibiting cholesteryl ester transfer protein (CETP). Two such agents have undergone large-scale clinical evaluation and have not shown efficacy in improving cardiovascular outcomes. Clinical studies currently under way will assess the effectiveness of two other CETP inhibitors that lack some of the adverse off-target actions encountered with the first agent tested.
TABLE 291e-3Heart Healthy Nutrition and Physical Activity Behaviors Recommended in the 2013 ACC/AHA Guideline on Lifestyle Management to Reduce Cardiovascular Risk ||Download (.pdf) TABLE 291e-3Heart Healthy Nutrition and Physical Activity Behaviors Recommended in the 2013 ACC/AHA Guideline on Lifestyle Management to Reduce Cardiovascular Risk
|The adult population should be encouraged to practice heart healthy lifestyle behaviors, including: |
Consume a dietary pattern that emphasizes intake of vegetables, fruits, and whole grains; include low-fat dairy products, poultry, fish, legumes, nontropical vegetable oils, and nuts; and limit intake of sodium, sweets, sugar-sweetened beverages, and red meats.
Adapt this dietary pattern to appropriate calorie requirements, personal and cultural food preferences, and nutrition therapy for other medical conditions (including diabetes mellitus).
Achieve this pattern by following plans such as the DASH dietary pattern, the USDA Food Pattern, or the AHA Diet.
Engage in 2 h and 30 min a week of moderate-intensity or 1 h and 15 min (75 min) a week of vigorous-intensity aerobic physical activity, or an equivalent combination of moderate- and vigorous-intensity aerobic physical activity. Aerobic activity should be performed in episodes of at least 10 min, preferably spread throughout the week.
Achieve and maintain a healthy weight. Refer to the 2013 Obesity Expert Panel Report for recommendations on weight loss and maintenance.
The mechanism by which elevated LDL levels promote atherogenesis may involve oxidative modification. Yet, rigorous and well-controlled clinical trials have failed to demonstrate that antioxidant vitamin therapy improves coronary heart disease (CHD) outcomes. In regard to nontraditional risk factors including homocysteine and infection, large-scale clinical trials using vitamins to lower homocysteine or using antibiotics have not reduced cardiovascular events. Therefore, the current evidence base does not support the use of vitamins or antibiotics to lower cardiovascular risk.
(See also Chap. 298) A wealth of epidemiologic data support a relationship between hypertension and atherosclerotic risk, and extensive clinical trial evidence has established that pharmacologic treatment of hypertension can reduce the risk of stroke, heart failure, and CHD events.
Diabetes Mellitus, Insulin Resistance, and the Metabolic Syndrome
(See also Chap. 417) Most patients with diabetes mellitus die of atherosclerosis and its complications. Aging and rampant obesity underlie a current epidemic of type 2 diabetes mellitus. The abnormal lipoprotein profile associated with insulin resistance, known as diabetic dyslipidemia, accounts for part of the elevated cardiovascular risk in patients with type 2 diabetes. Although diabetic individuals often have LDL cholesterol levels near the average, the LDL particles tend to be smaller and denser and, therefore, more atherogenic. Other features of diabetic dyslipidemia include low HDL and elevated triglyceride levels. Hypertension also frequently accompanies obesity, insulin resistance, and dyslipidemia. This commonly encountered clinical cluster of risk factors has become known as the metabolic syndrome (Chap. 422). Despite legitimate concerns about whether clustered components confer more risk than the individual components, the metabolic syndrome concept may offer clinical utility.
Therapeutic objectives for intervention in these patients include addressing the underlying causes, including obesity and low physical activity, by initiating lifestyle measures (see below). Establishing that strict glycemic control reduces the risk of macrovascular complications of diabetes has proved much more elusive than the beneficial effects on microvascular complications such as retinopathy and renal disease. Indeed, “tight” glycemic control may increase adverse events in patients with type 2 diabetes, lending even greater importance to aggressive control of other aspects of risk in this patient population. In this regard, multiple clinical trials have demonstrated unequivocal benefit of statin therapy in diabetic patients over all ranges of LDL cholesterol levels (but not those with end-stage renal disease or advanced heart failure). Among the oral hypoglycemic agents, metformin possesses the best evidence base for cardiovascular event reduction. The novel oral hypoglycemic agents tested in sufficiently powered trials, the dipeptidyl peptidase-4 (DPP-4) inhibitors saxagliptin and alogliptin, did not show cardiovascular benefit. Indeed, saxagliptin was associated with a slight increase in heart failure. Diabetic populations appear to derive particular benefit from antihypertensive strategies that block the action of angiotensin II. Thus, the antihypertensive regimen for patients with the metabolic syndrome should include angiotensin-converting enzyme inhibitors or angiotensin receptor blockers when possible. Many of these individuals will require more than one antihypertensive agent to reach the 2013 goals for individuals 18 years of age or older with diabetes to achieve a systolic blood pressure of less than 140 mmHg and a diastolic blood pressure of less than 90 mmHg.
Male Sex/Postmenopausal State
Decades of observational studies have verified excess coronary risk in men compared with premenopausal women. After menopause, however, coronary risk accelerates in women. Although observational and experimental studies have suggested that estrogen therapy reduces coronary risk, large-scale randomized clinical trials have not demonstrated a net benefit of estrogen with or without progestins on CHD outcomes. In the Heart and Estrogen/Progestin Replacement Study (HERS), postmenopausal female survivors of acute MI were randomized to an estrogen/progestin combination or to placebo. This study showed no overall reduction in recurrent coronary events in the active treatment arm. Indeed, early in the 5-year course of this trial, a trend occurred toward an increase in vascular events in the treated women. Extended follow-up of this cohort did not disclose an accrual of benefit in the treatment group. The Women’s Health Initiative (WHI) study arm, using a similar estrogen plus progesterone regimen, was halted due to a small but significant hazard of cardiovascular events, stroke, and breast cancer. The estrogen without progestin arm of WHI (conducted in women without a uterus) was stopped early due to an increase in strokes, and failed to afford protection from MI or CHD death during observation over 7 years. The excess cardiovascular events in these trials may result from an increase in thromboembolism (Chap. 413). Physicians should work with women to provide information and help weigh the small but evident CHD risk of estrogen ± progestin versus the benefits for postmenopausal symptoms and osteoporosis, taking personal preferences into account. Post hoc analyses of observational studies suggest that estrogen therapy in women younger than or closer to menopause than the women enrolled in WHI might confer cardiovascular benefit. Thus, the timing in relation to menopause or the age at which estrogen therapy begins may influence its risk/benefit balance.
The lack of efficacy of estrogen therapy in cardiovascular risk reduction highlights the need for redoubled attention to known modifiable risk factors in women. Meta-analysis supports the efficacy of statins to reduce cardiovascular events in women in primary prevention, as well as in those who have already experienced a cardiovascular event.
Dysregulated Coagulation or Fibrinolysis
Thrombosis ultimately causes the gravest complications of atherosclerosis. The propensity to form thrombi and/or lyse clots once they form influences the manifestations of atherosclerosis. Thrombosis provoked by atheroma rupture and subsequent healing may promote plaque growth. Certain individual characteristics can influence thrombosis or fibrinolysis and have received attention as potential coronary risk factors. For example, fibrinogen levels correlate with coronary risk and provide information about coronary risk independent of the lipoprotein profile.
The stability of an arterial thrombus depends on the balance between fibrinolytic factors, such as plasmin, and inhibitors of the fibrinolytic system, such as plasminogen activator inhibitor 1 (PAI-1). Individuals with diabetes mellitus or the metabolic syndrome have elevated levels of PAI-1 in plasma, and this probably contributes to the increased risk of thrombotic events. Lp(a) (Chap. 421) may modulate fibrinolysis, and individuals with elevated Lp(a) levels have increased CHD risk.
Aspirin reduces CHD events in several contexts. Chapter 293 discusses aspirin therapy in stable ischemic heart disease, Chap. 294 reviews recommendations for aspirin treatment in acute coronary syndromes, and Chap. 446 describes aspirin’s role in preventing recurrent ischemic stroke. In primary prevention, pooled trial data show that low-dose aspirin treatment (81 mg/d to 325 mg on alternate days) can reduce the risk of a first MI in men. Although the Women’s Health Study (WHS) showed that aspirin (100 mg on alternate days) reduced strokes by 17%, it did not prevent MI in women. Current AHA guidelines recommend the use of low-dose aspirin (75–160 mg/d) for women with high cardiovascular risk (≥20% 10-year risk), for men with a ≥10% 10-year risk of CHD, and for all aspirin-tolerant patients with established cardiovascular disease who lack contraindications.
An accumulation of clinical evidence shows that markers of inflammation correlate with coronary risk. For example, plasma levels of CRP, as measured by a high-sensitivity assay (hsCRP), prospectively predict the risk of MI. CRP levels also correlate with the outcome in patients with acute coronary syndromes. In contrast to several other novel risk factors, CRP adds predictive information to that derived from established risk factors, such as those included in the Framingham score (Fig. 291e-6). Mendelian randomization studies do not support a causal role for CRP in cardiovascular disease. Thus, CRP serves as a validated biomarker of risk, but probably not as a direct contributor to pathogenesis.
C-reactive protein (CRP) level adds to the predictive value of the Framingham score. hsCRP, high-sensitivity measurement of CRP. (Adapted from PM Ridker et al: Circulation 109:2818, 2004.)
Elevations in acute-phase reactants such as fibrinogen and CRP reflect the overall inflammatory burden, not just vascular foci of inflammation. Visceral adipose tissue releases proinflammatory cytokines that drive CRP production and may represent a major extravascular stimulus to the elevation of inflammatory markers in obese and overweight individuals. Indeed, CRP levels rise with body mass index (BMI) or visceral adipose depot as assessed by imaging, and weight reduction lowers CRP levels. Infectious agents might also furnish inflammatory stimuli related to cardiovascular risk.
Statin therapy likely reduces cardiovascular events in part by muting the inflammatory aspects of the pathogenesis of atherosclerosis. For example, in statin trials conducted in both primary (JUPITER) and secondary (PROVE-IT/TIMI-22) prevention populations, prespecified analyses showed that those who achieved lower levels of both LDL and CRP had better clinical outcomes than did those who only reached the lower level of either the inflammatory marker or the atherogenic lipoprotein (Fig. 291e-7). The anti-inflammatory effect of statins appears independent of LDL lowering, because these two variables correlated very poorly in individual subjects in multiple clinical trials.
Evidence from the JUPITER study that both low-density lipoprotein (LDL)-lowering and anti-inflammatory actions contribute to the benefit of statin therapy in primary prevention. See text for explanation. hsCRP, high-sensitivity measurement of C-reactive protein (CRP). (Adapted from PM Ridker et al: Lancet 373:1175, 2009.)
The prevention of atherosclerosis presents a long-term challenge to all health care professionals and for public health policy. Both individual practitioners and organizations providing health care should strive to help patients optimize their risk factor profiles long before atherosclerotic disease becomes manifest. The current accumulation of cardiovascular risk in youth and in certain minority populations presents a particularly vexing concern from a public health perspective.
The ACC/AHA 2013 Guideline on Lifestyle Management to Reduce Cardiovascular Risk relied on rigorous evidentiary reviews. Few lifestyle interventions have undergone rigorous evaluation in randomized clinical trials. Therefore, these guidelines reflected judicious analysis of carefully selected observational studies and of intervention studies that relied primarily on biomarkers or surrogate endpoints rather than “hard” cardiovascular outcomes. Table 291e-3 summarizes the ACC/AHA lifestyle recommendations.
The care plan for all patients seen by internists should include measures to assess and minimize cardiovascular risk. Physicians must counsel patients about the health risks of tobacco use and provide guidance and resources regarding smoking cessation. Similarly, physicians should advise all patients about prudent dietary and physical activity habits for maintaining ideal body weight. Both National Institutes of Health (NIH) and AHA statements recommend at least 30 min of moderate-intensity physical activity per day. Obesity, particularly the male pattern of centripetal or visceral fat accumulation, can contribute to the elements of the “metabolic syndrome” cluster. Physicians should encourage their patients to take personal responsibility for behavior related to modifiable risk factors for the development of premature atherosclerotic disease. Conscientious counseling and patient education may forestall the need for pharmacologic measures intended to reduce coronary risk.
Issues in Risk Assessment
A growing panel of markers of coronary risk presents a perplexing array to the practitioner. Markers measured in peripheral blood include size fractions of LDL particles and concentrations of homocysteine, Lp(a), fibrinogen, CRP, PAI-1, myeloperoxidase, lipoprotein-associated phospholipase A2, and imaging assessment of subclinical atherosclerosis, among many others. In general, such specialized tests add little to the information available from a careful history and physical examination combined with measurement of a plasma lipoprotein profile and fasting blood glucose. The hsCRP measurement may well prove an exception in view of its robustness in risk prediction, ease of reproducible and standardized measurement, relative stability in individuals over time, ability to add to the risk information disclosed by standard measurements such as the components of the Framingham risk score, and most importantly, the demonstration in a large-scale trial (JUPITER) that allocating therapy can reduce cardiovascular events in those deemed ineligible by traditional risk assessment criteria. The addition of information regarding a family history of premature atherosclerosis (a simply obtained indicator of genetic susceptibility), together with the inflammation marker hsCRP, permits correct reclassification of risk in individuals, especially those whose Framingham scores place them at intermediate risk.
Available data do not support the routine use of imaging studies to screen for subclinical disease (e.g., measurement of carotid intima-media thickness, coronary artery calcification, and use of computed tomographic coronary angiograms [CTA]). Inappropriate use of such imaging modalities may promote excessive alarm in asymptomatic individuals and prompt invasive diagnostic and therapeutic procedures of unproven value for both asymptomatic atherosclerosis and incidental findings. Widespread application of such modalities for screening should await proof that targeting therapies based on their application provides clinical benefit.
The 2013 ACC/AHA Guideline on the Assessment of Cardiovascular Risk recommends the use of newer risk markers if uncertainty persists after assessing quantitative risk using the pooled cohort calculator. The guideline states that family history, hsCRP, coronary artery calcium (CAC) score, or ankle-brachial index (ABI) may then be considered to inform treatment decision making. It discourages carotid intima-media thickness (CIMT) for routine measurement in clinical practice for risk assessment for a first ASCVD event. The guideline panel deemed the contribution to risk assessment for a first ASCVD event using apolipoprotein B (ApoB), chronic kidney disease, albuminuria, or cardiorespiratory fitness as uncertain at present.
Progress in human genetics holds considerable promise for risk prediction and for individualization of cardiovascular therapy. Many early reports identified single-nucleotide polymorphisms (SNPs) in candidate genes as predictors of cardiovascular risk. The validation of such genetic markers of risk and drug responsiveness in multiple populations often proved disappointing. The era of GWAS has led to discovery of sites of genetic variation that reproducibly indicate heightened cardiovascular risk (e.g., chromosome 9p21). The advent of technology that permits relatively rapid and inexpensive exome or whole-genome sequencing promises to identify new therapeutic targets, sharpen risk prediction, and deploy preventive or therapeutic measures in a more personalized manner. Despite this considerable promise, genetic scores for risk prediction have not yet demonstrated consistent improvement over algorithms that use traditional tools.