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In Nature Medicine, November 2002, Volume 8 Number 11 pp 1211 - 1217, a section about atherosclerosis was introduced by Daniel Steinberg: Atherogenesis in perspective: Hypercholesterolemia and inflammation as partners in crime. Here is a letter, commenting Steinbergs article by Uffe Ravnskov. His covering letter follows as does the answer from the editor. Steinbergs paper is available below. 

Uffe Ravnskov: High cholesterol does not cause atherosclerosis

According to Daniel Steinberg  “there is no longer a cholesterol controversy” (November 2002,  p 1211). Actually the opposite is true: the cholesterol hypothesis can no longer stand against a large body of contradictory findings. For example, there is no correlation between blood cholesterol and degree of atherosclerosis at autopsy. Investigation by angiography reveals little or no association. Electron beam tomography, a new technique that allows the identification of intramural deposits, reveals an inverse correlation between LDL cholesterol and degree of coronary calcification. Most importantly, a large number of observational and experimental angiographic studies have failed to reveal exposure-response between the changes of total or LDL-cholesterol and changes of coronary atherosclerosis, and clinical trials have failed to find exposure-response between degree of cholesterol lowering and outcome.1,2

   Steinberg also claims that high cholesterol is sufficient in itself to produce atherosclerosis, referring to reports of early cardiovascular disease in individuals with familial hypercholesterolaemia (FH). But these patients have been selected because of disease.  A recent study of unselected individuals with FH traced backwards found that before year 1900 their mortality was lower than that of the general population.3 Also, if hypercholesterolaemia is “a sine qua non for…. clinically important atherosclerosis”, it is difficult to explain why low, not high cholesterol is a risk factor for coronary heart disease for people above age 70, the period of life where the great majority of cardiovascular events occur.1,2

     Steinberg joins many other apologists for the diet-heart theory in claiming that animal experiments prove that high cholesterol causes heart disease. The animals used most frequently in these experiments are vegetarian rabbits and mice, simply because omnivorous or carnivorous animals show small or no vascular effects when their cholesterol is raised by diet. But how do we know whether human beings react to high cholesterol as the herbivorous rabbit or as the omnivorous rat? 

     Furthermore, no animal study has ever produced a heart attack solely by raising an animal’s cholesterol. What these studies have produced is fatty streaks, not atherosclerosis. According to Steinberg there is consensus regarding the sequence of events from hypercholesterolaemia to fatty streaks. The crucial question however is, whether fatty streaks develop into true atherosclerosis. Steinberg admits that there is no hypothesis to explain that development. This is no surprise because fatty streaks are seen equally often in populations where atherosclerosis is rare as in populations where it is prevalent,4 clearly indicating that fatty streaks are not the very forerunners of atherosclerosis. A link is missing. 

  1. Ravnskov U. Is atherosclerosis caused by high cholesterol? Q. J. Med.  95: 397-403 (2002).
  2. Ravnskov U. A hypothesis out-of-date: The diet-heart idea. J Clin Epidemiol. 2002 Nov;55(11):1057-63.
  3. Eric J G Sijbrands E.J.G. et al. Mortality Over Two Centuries in Large Pedigree With Familial Hypercholesterolaemia: Family Tree Mortality Study BMJ 322, 1019-1023  (2001).
  4. Strong J.P., Eggen D.A., Oalmann MC, Richards M.L., & Tracy RE. Pathology and epidemiology of atherosclerosis. J. Am. Diet. Assoc. 62, 262-268 (1973).

Uffe Ravnskov's covering letter:

Editor

With great surprise I read the introduction to your ”Special Focus on Atherosclerosis” by Daniel Steinberg. In the attached letter-to-the-editor I have pointed to a few of the numerous erroneous and misleading statements from Dr. Steinberg. However, with a limit of 400 words it is impossible to expose the full width of Dr. Steinbergs disingenuous article. In addition to the objections in my letter I have therefore given a more detailed criticism as follows.

The first misleading or ambiguous statement said that “…correcting hypercholesterolemia profoundly reduces morbidity and mortality …from CHD”, referring to the statin trials. Apart from the fact that Steinberg, like most other proponents of the cholesterol campaign, ignores the first, large statin trial, EXCEL, where total mortality increased by 150 % (0,5 % in the Lovastatin group, 0.2 % in the control group) already after one year,1 there is much evidence that the benefit exerted by the statins has nothing to do with their effect on cholesterol metabolism. This is most evident from the lack of exposure-response, as I have also mentioned in my letter. I would like to cite the authors of the CARE study2:

 “In a multivariate analysis that included LDL concentration during follow-up, the change in LDL from baseline, expressed either as a percentage or absolute change in concentration, was not found to be significantly related to coronary events*.”

(* Indeed, because p was 0.97 for absolute change and 0.76 for percentage change; my comment)

 In addition, in a recent paper I analysed this problem in all angiographic studies, where exposure-response had been calculated, a total of five observational studies and 18 trials. Except for one trial no study found exposure-response; indeed, in two of the observational studies an inverse correlation was found between changes of cholesterol and changes of angiographic atherosclerosis.3 

In the second section (Hypercholesterolemia as the initiator of atherosclerosis) Steinberg claimed that “the benefit (of the statins) relates to the change in cholesterol in much the same way whether the cholesterol lowering is achieved with diet or with drugs.” Apart from the fact, mentioned above, that the benefit was not associated with the change in cholesterol, the accumulated evidence from all controlled, randomised, unifactorial dietary trials did not find any effect on coronary or total mortality; in fact, the total number of deaths in the diet and the control arms were identical. This was shown by me in a previous review4 and confirmed last year by Hooper et al. in BMJ.5,6

Steinberg continues misquoting by claiming that “drugs that lower cholesterol levels by different mechanisms have conferred benefit predicted by the extent to which cholesterol levels have been reduced. “ Not only has a meta-analysis of the non-statin trials shown that coronary mortality was unchanged after cholesterol lowering and total mortality increased, but also that the outcome was independent of the degree of cholesterol lowering, both within each trial but also between the trials.7

 In the same section the reader is told that the risk for CHD is “a smooth, continuous function of cholesterol level”. Nothing could be further from the truth. In women, the risk is trivial. A review of eleven cohort studies, including more than 120,000 women, found an increased risk for coronary mortality in the fourth cholesterol quartile only (RR 1.56), whereas the risk for all cardiovascular and for total mortality was independent on the cholesterol level.8 In men, there are many populations where a high cholesterol level is not predictive of CHD. In Framingham, for example, a decreasing cholesterol level predicted an increased risk of coronary and total mortality. For each 1 mg/dl drop of cholesterol there was an 11 percent increase in coronary and total mortality.9  In Russia low cholesterol predicts higher coronary and total mortality.10 And as mentioned in my letter, high cholesterol does not predict CHD in the elderly either.11-13 Indeed, the higher the cholesterol level, the lower the risk.12,13

There is increasing evidence that inflammatory processes in the arterial walls, initiated by various factors, for instance microorganisms, homocysteine, toxic chemicals and/or free radicals), are the starting point for the production of raised lesions. Steinberg’s article is a typical post hoc attempt to incorporate the cholesterol hypothesis with the new ideas, although all evidence has shown that high cholesterol is a biological marker, an innocent bystander without influence itself on the pathological processes.

In my letter-to-the-editor I have referred to a paper in print . I have attached that manuscript (not corrected for linguistic errors). The editor asked Professor William S. Weintraub at Emory University, Atlanta, USA, a well-known supporter of the cholesterol campaign, to comment my review, and he also asked me to comment Professor Weintraub’s comment. I have therefore also attached Professor Weintraub’s dissent and my comment to his dissent.

Yours sincerely 

Uffe Ravnskov, MD, PhD, independent researcher
Magle Stora Kyrkogata 9, S-22350 Lund, Sweden 
Homepage:  http://www.ravnskov.nu/uffe.htm

  1. Bradford RH et al.. Expanded Clinical Evaluation of Lovastatin (EXCEL) study results. I. Efficacy in modifying plasma lipoproteins and adverse event profile in 8245 patients with moderate hypercholesterolemia. Arch. Intern. Med. 151,43-49 (1991)
  2. Sacks FM et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators. N Engl J Med 335, 1001-1009 (1996)
  3. Ravnskov U. Is atherosclerosis caused by high cholesterol? Quart. J. Med.  95, 397-403 (2002). This paper was also selected for publication in the third issue of South African Excerpts Edition of the QJM 2002. The full paper is available on http://qjmed.oupjournals.org/cgi/content/full/95/6/397?ijkey=/Uh9j66HOHg.Y
  4. Ravnskov U. The questionable role of saturated and polyunsaturated fatty acids in cardiovascular disease. J. Clin. Epidemiol. 51:443-460 (1998).   This paper won the Skrabanek Award 1999.
  5. Hooper, L. et al. Dietary Fat Intake And Prevention of Cardiovascular Disease: Systematic Review BMJ 322, 757-763 (2001).
  6. Ravnskov U. Diet-heart disease hypothesis is wishful thinking. BMJ 324: 238 (2002)
  7. Ravnskov U. Cholesterol lowering trials in coronary heart disease: frequency of citation and outcome. BMJ 305, 15-19 (1992).
  8. Jacobs D. et al. Report of the conference on low blood cholesterol: Mortality associations. Circulation 86, 1046-1060 (1992)
  9. Anderson K.M., Castelli W.P. & Levy D. Cholesterol and mortality. 30 years of follow-up from the Framingham study. JAMA 257, 2176-2180 (1987).
  10. Shestov D.B., Deev A.D., Klimov A.N., Davis C.E., Tyroler H.A.. Increased risk of coronary heart disease death in men with low total and low-density lipoprotein cholesterol in the Russian Lipid Research Clinics Prevalence Follow-up Study. Circulation 88, 846-853 (1993)
  11. Zimetbaum P et al. Plasma lipids and lipoproteins and the incidence of cardiovascular disease in the very elderly. The Bronx aging study. Arterioscl Thromb 1992; 12, 416-423 (1992).
  12. Krumholz H.M. et al. Lack of association between cholesterol and coronary heart disease mortality and morbidity and all-cause mortality in persons older than 70 years. JAMA 272: 1335-1340 (1994).
  13. Schatz IJ et al. Cholesterol and all-cause mortality in elderly people from the Honolulu Heart program: a cohort study. Lancet 358: 351-355 (2001)

 Editors answer:

December 19, 2002  
  
Our ref: NMED-LE15985  
  
Dear Dr. Ravnskov,  
  
Thank you for submitting your letter, "High cholesterol does not cause atherosclerosis", (NMED-LE15985), for possible publication in Nature Medicine. We have given the work our careful consideration, and decided that we are unable to offer to publish the letter.  
  
The space available for publishing such letters in Nature Medicine is very limited and in light of the many that are submitted, we must decline to publish the vast majority of them. In deciding which to publish we consider how topical each piece is, whether it is likely to be of interest to a broad selection of our readers and how well presented it is.    
  
In this case, the Steinberg piece was meant to be opinionated, and hence was labeled as a "perspective." We will continue to debate the issues of hypercholesterolemia vs inflammation in our research section, but do not feel any further discussion is warranted in the Letters or Reviews section. I am sorry that we cannot be more positive on this occasion and hope that this decision will not dissuade you from writing to us again.  
  
Sincerely yours,  
  
  
Ushma Savla, Ph.D.
Assistant Editor
Nature Medicine  

Daniel Steinbergs paper

Special Focus on Atherosclerosis
doi:10.1038/nm1102-1211
November 2002 Volume 8 Number 11 pp 1211 - 1217
  
Atherogenesis in perspective: Hypercholesterolemia and inflammation as partners in crime
Daniel Steinberg
 Department of Medicine University of California San Diego, La Jolla, California, USA e-mail: dsteinberg@ucsd.edu

 A historical perspective on atherosclerosis allows us to reflect on the once controversial hypotheses in the field. Plaque formation was once thought to be dependent upon hypercholesterolemia alone, or solely in response to injury. More recently, inflammatory cascades were thought to be at the root of lesion development. A more realistic view may be that atherosclerosis is neither exclusively an inflammatory disease nor solely a lipid disorder: it is both.

Atherosclerosis research entered a new era at the turn of the century. The twentieth century was the era of cholesterol and lipoproteins, culminating in a series of large-scale clinical trials showing conclusively that correcting hypercholesterolemia profoundly reduces morbidity and mortality from disease of the coronary arteries of the heart (coronary heart disease; CHD). In individuals at moderate to high risk for CHD, intensive treatment with statins (specific inhibitors of endogenous cholesterol biosynthesis) reduces plasma low-density lipoprotein (LDL) cholesterol (Fig. 1) levels by 30–50% and reduces CHD mortality to about the same extent over a 5-year period1-6. Notably, total mortality is also reduced: that is, lowering cholesterol levels does not, as some have maintained, increase the risk of non-cardiovascular causes of death. If treatment were started earlier in life and continued for longer times, these already remarkable effects would be greater still. Many countries have already instituted broad public health programs to reduce blood cholesterol levels. In short, the 'lipid hypothesis' of atherosclerosis has been validated and there is no longer a 'cholesterol controversy'7.

 Beginning about 20 years ago, and accelerating swiftly since then, research efforts have been increasingly focused on the pathobiology of the artery wall and the involvement of inflammation. How does the artery respond to hypercholesterolemia and other potential initiators of atherosclerosis? Vascular biologists8 have been actively exploring the complex interactions between the resident cells of the artery wall and the invading monocytes and T cells (the inflammatory processes), the nature of the cytokines and growth factors involved and the effects of these cell–cell interactions on rates of lesion development. An understanding of the complex inflammatory responses should allow the identification of key processes that could then be targeted to inhibit progression of the disease and its complications. These new initiatives have already yielded valuable insights and indicated new directions9.

There has been an unfortunate tendency to consider atherosclerosis as being either a lipid disorder or an inflammatory disorder. This is a false dichotomy. One purpose of this review is to emphasize that it is both, that they are two facets of a single pathogenesis. Indeed, atherosclerosis as an inflammatory disease is initiated by and progresses in the context of hypercholesterolemia.

Hypercholesterolemia as the initiator of atherosclerosis
The causal relationship between blood cholesterol and atherosclerosis is no longer in doubt7, 10, 11. The pooled results of many trials using different cholesterol-lowering regimens indicate that for every 10% reduction in cholesterol level, CHD deaths will be reduced by at least 15% (ref. 12). Today we know that lowering LDL levels benefits both men and women, old as well as young, diabetics as well as non-diabetics, and those with very high LDL levels as well as those with initial LDL levels as low as 100 mg/dl (ref. 6), once considered the target level of the most aggressive therapy13.

It has been assumed that the reduction in adverse clinical events when plasma cholesterol levels are decreased is directly related to the magnitude of the cholesterol lowering. That assumption is supported by the fact that the benefit relates to the change in cholesterol level in much the same way whether the cholesterol lowering is achieved with diet or with drugs12. Moreover, drugs that lower cholesterol levels by different mechanisms have conferred benefit predicted by the extent to which cholesterol levels have been reduced. However, recent studies have shown that statins, in addition to decreasing LDL levels, may exert anti-atherosclerotic effects by other mechanisms. These 'pleiotropic' effects, probably related to inhibition of the production of other isoprenoids such as geranylgeraniol or ubiquinone, are well-established in vitro, and studies are beginning to show cholesterol-independent mechanisms, including anti-inflammatory effects, in vivo as well14, 15. The extent to which these LDL-independent effects contribute to the clinical effects of the statins in humans has not yet been established16, 17.

Hypercholesterolemia: How high is 'high'?
A principal factor that held up progress was a misconception about how to define 'normal' in the case of blood cholesterol. The almost universal practice in clinical medicine was (and still is) to measure a parameter such as blood sodium in a large number of healthy subjects, and then define any value above the 95th percentile as abnormal. By that criterion, any blood cholesterol below 300 mg/dl would have been (and indeed was) considered to be 'normal'. Now we know that a blood cholesterol of even 240 mg/dl is much too high, as it is associated with substantially increased risk of CHD, and we know that that risk can be sharply reduced by lowering the cholesterol level. The unappreciated point is that the cholesterol levels in much of the population of Western countries are unhealthily high; that is, the distribution curve for the entire ('normal') population is shifted too far to the right. Of course, there is no sharply defined threshold or cutoff point; risk, all else being equal, is a smooth, continuous function of cholesterol level (although the slope of the curve is higher at higher levels).

Hypercholesterolemia: Is it ever a sufficient cause of CHD?
The notably premature CHD in patients with familial hypercholesterolemia (as early as 5 years of age) strongly indicated that a very high cholesterol level, especially a high LDL-cholesterol level, was in itself enough to account for severe atherosclerotic disease. These children have no other risk factors: They do not smoke, do not have hypertension, are not obese, and so on. However, it was still possible that the abnormal gene involved might independently lead to hypercholesterolemia by one pathway and to premature vascular disease by some unrelated pathway. The identification of the defective gene as being the gene for the LDL receptor (LDLR)18 established that the pathogenetic sequence of events was that a defective LDLR leads to high plasma LDL, which in turn leads to atherosclerosis. Thus, hypercholesterolemia can be a sufficient cause.

Hypercholesterolemia: Is it a necessary cause of CHD?
The rate at which atherosclerosis progresses can vary considerably at any given plasma cholesterol level. For example, siblings with familial hypercholesterolemia may have identical cholesterol levels and yet one may have a fatal myocardial infarction at age 10 and the other survive to age 50 (ref. 19). Clearly, there are additional factors determining just how fast the disease progresses. Indeed, atherosclerosis is a multifactorial disease of great complexity, and we are only now beginning to sort out the many elements involved, especially in the later stages of the disease. However, it may be an informative exercise to entertain the possibility that some degree of hypercholesterolemia is a sine qua non for the expression of clinically important atherosclerosis. Thus, lesion formation may be driven primarily (but by no means exclusively) by hypercholesterolemia, whereas the other risk factors, local and systemic, exert their influence by affecting an underlying lipoprotein-driven pathogenesis.

Whether hypercholesterolemia qualifies as a necessary cause depends on how 'high' cholesterol is defined. Humans are unique in having LDL cholesterol levels much higher than those of any other mammalian species and are also unique in spontaneously developing atherosclerosis and CHD (ref. 20). The mean LDL level in lower animals averages 32 mg/dl (ref. 21). If humans had LDL levels of less than 32 mg/dl, would they also be free of clinically relevant atherosclerotic disease? It is possible. The CHD death rate in Japan 50 years ago, when the total cholesterol levels of the Japanese averaged about 160 mg/dl and their LDL levels were less than 100 mg/dl, was about one-tenth that in the US. This was true despite the fact that the prevalence of hypertension and cigarette smoking, two of the principal risk factors for CHD, was higher in Japan than in the US. Is it possible that these principal risk factors influence the rate of progression of atherosclerosis substantially only when the plasma level of LDL is increased above at least some 'threshold' level? Although low HDL is a chief risk factor for atherosclerosis, knocking out the HDL apolipoprotein AI gene in the mouse, although it decreases HDL levels substantially, does not by itself induce atherosclerotic lesions22. Only when the accompanying LDL levels are increased, as in the apolipoprotein E–deficient mouse, does the pro-atherogenic potential of the low HDL become apparent23.

Thus, when considering pathogenesis, it is important to remember that the best documented initiating factor in atherogenesis is still hypercholesterolemia and that additional factors, of which there are many, should whenever possible be considered in the context of how they relate to the processes initiated by hypercholesterolemia.

How is blood cholesterol involved in pathogenesis?
There is now a consensus regarding the sequence of events triggered by hypercholesterolemia and leading to the generation of the earliest visible lesion of atherosclerosis, the fatty streak lesion (Fig. 2). Much of the early work leading to this scheme came from studies in the hypercholesterolemic rabbit, but for a decade now the preferred model has been the mouse. The C57Bl/6J mouse was first proposed as a model in 1987 (ref. 24), but because its lesions were limited to the aortic valve region most investigators doubted the validity of the model. However, with the introduction of the apolipoprotein E–deficient mouse25 and the LDLR-deficient mouse26, this skepticism was overcome and the mouse has become the preferred animal model. It is now possible to capitalize on the well-characterized inbred mouse lines and, most importantly, to apply gene-targeting techniques. Gene 'knockouts' and 'knock-ins' are now the gold standard for critically testing the relevance of candidate genes in atherogenesis.

One of the very earliest responses induced by hypercholesterolemia is an increase in the expression of vascular cell-adhesion molecule 1, a key adhesion molecule for monocytes and T cells, on the endothelial surface lining the major arteries27. The functional importance of this adhesion molecule has been demonstrated by knockout studies in mouse models28. Knockout studies have also indicated involvement of P- and E-selectins29 and intracellular adhesion molecule 1 (ref. 30). Hypercholesterolemia also induces an increase in the expression of monocyte chemoattractant protein 1 (MCP-1), a key chemotactic factor in the artery wall31, and increases the expression of its receptor on monocytes32. Again, the best evidence for the essential involvement of MCP-1 and its receptor in atherogenesis comes from gene-targeting studies in mouse models33, 34. Other adhesion molecules and other chemokines, such as interleukin 8 (ref. 35), may be involved as well. The net result is the recruitment of monocytes (and later T lymphocytes) to the sites of developing lesions, where they penetrate and take up residence in the subendothelial space. There, the monocytes undergo phenotypic modification and take up LDL to become foam cells, loaded with multiple cytoplasmic droplets containing cholesterol esters.

Pathogenesis: The foam-cell paradox
The cholesterol accumulating in foam cells is known to be ultimately derived from circulating plasma lipoproteins. However, incubation of neither monocytes and macrophages nor arterial smooth muscle cells with native LDL, even at very high concentrations, increases their cell cholesterol content substantially36, 37. These cells, like other cells, protect themselves against cholesterol overloading by downregulating the native LDLR (Fig. 3). LDL must be somehow modified before it can induce foam-cell formation37. A very effective modification was achieved by chemical acetylation of LDL by treatment with acetic anhydride in vitro. In contrast to native LDL, acetyl LDL was taken up very rapidly by macrophages and caused massive cholesterol accumulation. Moreover, uptake was not through the native LDLR but through another receptor called the 'acetyl LDLR'. The receptor was later cloned and renamed scavenger receptor A (SR-A)38. Unlike LDLR, SR-A does not downregulate in response to an increase in cellular cholesterol content and this could, in theory, account for foam-cell formation. However, there was and still is no evidence that acetyl LDL is ever generated in vivo, and so the search for modified forms of LDL continued. A review article summarizing studies of macrophage handling of lipoproteins described many other modified forms of LDL that might account for foam-cell formation39. None of those has been shown to be involved in vivo, but not all have been subjected to careful study. The best studied modification that might account for foam-cell formation is oxidative modification40-42. Oxidized LDL (oxLDL), whether generated by incubation with cells in culture or by incubation with copper as a catalyst, is taken up avidly by macrophages and can cause foam-cell formation (Fig. 3). It is taken up in part by way of SR-A but to an even greater extent by way of other scavenger receptors, especially CD36 (ref. 43). Circulating monocytes express SR-A and CD36 at fairly low levels, but when the monocyte enters the artery wall, it undergoes a series of phenotypic modulations and becomes an arterial tissue macrophage. One of the important phenotypic changes is an increase in the expression of scavenger receptors, which, as discussed above, account for the ability of the macrophage to take up modified forms of LDL. Some foam cells are also derived from smooth muscle cells, probably because these cells can express scavenger receptors when appropriately activated44. 

Pathogenesis: The oxidative modification hypothesis
Presumably the rate of production of oxLDL in the arterial intima is a function of the concentration of native LDL present. That concentration is proportional to the plasma LDL concentration45. Aortic wall concentrations of LDL in the rabbit are higher at lesion-susceptible sites than at lesion-resistant sites even before lesions appear46. However, endothelial permeability to LDL is not increased at those sites; thus, LDL is somehow selectively retained. This retention probably occurs because LDL adheres tightly to arterial wall proteoglycans47. Moreover, LDL bound to proteoglycans is more readily oxidized48. Thus, the proposition that oxidation of trapped LDL may be the initiating factor at susceptible sites is reasonable. It is further supported by the demonstration that LDL genetically engineered so that it does not bind to proteoglycans is less atherogenic than native LDL (ref. 49). These ideas have been expanded and extended50.

Oxidation of LDL was originally thought to be involved in pathogenesis because this could account for the loading of macrophages with cholesterol, but it quickly became apparent that oxLDL had many other properties that were potentially pro-atherogenic. For example, oxLDL is itself directly chemotactic for monocytes and T cells (but not for B cells or neutrophils, neither of which are found in lesions)51, 52. Among other biological effects, oxLDL (and its various oxidized lipid components) are cytotoxic for endothelial cells53, are mitogenic for macrophages and smooth muscle cells54, 55 and stimulate the release of monocyte chemoattractant protein 1 and of monocyte colony-stimulating factor from endothelial cells54, 55. The oxidative modification hypothesis has been extensively reviewed58-60.

Recently there has been considerable progress in identifying the components of oxLDL that make it a ligand for scavenger receptors61-64. Extensive degradation of the polyunsaturated fatty acid in the sn-2 position of phospholipids by oxidation seems to be essential. Moreover, oxLDL and apoptotic cells compete for binding to macrophage scavenger receptors65, 66, indicating that oxidized phospholipids in the membranes of apoptotic cells are involved in their binding to macrophage scavenger receptors.

That mice deficient in paraoxonase, an enzyme that degrades organophosphates, are more susceptible to atherosclerosis67 is also compatible with the idea that oxidized phospholipids are important in pathogenesis. The possibility that paraoxonase may be involved in the human disease is indicated by the fact that certain polymorphisms in the enzyme are associated with increased CHD risk; however, only preliminary studies have so far been done in this area68.

A considerable body of evidence supports the notion that oxLDL is involved in atherogenesis, but the most notable piece of evidence is the fact that several structurally distinct antioxidants have been shown to inhibit atherogenesis by 30–70% in different animal models58. However, it is not apparent that this hypothesis is relevant to the disease in humans. Initial clinical trials of antioxidant vitamins, most using vitamin E, have had mainly negative results; these include the recently reported Oxford Heart Protection Study6. These results may indicate only that the wrong antioxidants have been tested, at the wrong doses or at the wrong stage in disease development, but they do not, at least for now, refute the oxidative modification hypothesis69.

Pathogenesis: Do other modifications produce foam cells?
Aggregated LDL is taken up by macrophages more avidly than native LDL (ref. 70), apparently by a phagocytic pathway. LDL is well known to self-aggregate after it is removed from the plasma, and it aggregates under the right conditions within the artery wall71. Enzymatically modified LDL also can aggregate and be taken up more rapidly72. Immune complexes of LDL with immunoglobulins can enter the macrophage by means of the Fc receptor, and this might cause cholesterol accumulation, particularly if the LDL binding to the antibody is already aggregated73. These alternative modifications of LDL that might contribute to foam-cell formation should be studied further to determine whether they function under in vivo conditions.

Atherosclerosis as an inflammatory disease
Studies over the last two decades have demonstrated the many complex ways in which monocytes, macrophages and T cells in the developing lesion interact with each other and with the indigenous arterial endothelial and smooth muscle cells. Progress in this area has been summarized in several reviews8, 9, 74, 75 and in the review by Witztum in this focus. Therefore, this review will not provide details of this research, except to point out that atherosclerosis is properly regarded as an inflammatory process in which leukocytes enter the affected tissue site and are central to pathogenesis.

However, understanding what instigates arterial wall inflammation is necessary. Inflammation is a response to a disturbance in tissue or organ homeostasis. The classic example is infection, in which the response is to invading microorganisms. Another example is gouty arthritis, in which the response is to the formation of crystals of monosodium urate in the joint space: Initially the participation of the leukocytes may be beneficial, but ultimately the inflammatory process can cause excessive tissue damage and result in clinical gout. Inflammation occurs in response to something that destabilizes local homeostasis; in atherosclerosis, identification of that 'something' has proven elusive. Atherosclerosis has been called a 'response to injury', and initially the 'injury' was believed to consist of a loss of endothelial cells from the lining of the vessel74, 75. Later studies, however, showed that the endothelial monolayer overlying the earliest lesions was actually intact76. The injury or destabilizing factor that initiated the inflammatory process remained unknown. Candidates included oxLDL, mechanical injury, immunological injury, homocysteine and viruses8. As discussed below, the many biological effects of oxLDL are certainly compatible with its having an initiating function. The other candidates may be, and probably are, involved in atherogenesis, but there is little evidence that they can initiate atherogenesis in the absence of hypercholesterolemia. Much of the new evidence regarding inflammatory factors is derived from studies in mouse models of atherosclerosis and therefore is almost always derived in the context of extremely high levels of blood cholesterol. There is no doubt, however, that the superimposition of, for example, a viral injury or homocysteinemia on a cholesterol-induced lesion could make the difference between lesion progression that never leads to clinical CHD and lesion progression that kills. These and other non-lipid risk factors are deservedly receiving increasing attention, but each will require prospective intervention studies for ultimate validation as causative factors.

The pathway from fatty streaks to late lesions
The fatty streak is itself clinically benign, but it is the precursor to all the late, clinically relevant lesions. Preventing fatty-streak initiation would block formation of the stenotic fibrous plaque or the complex unstable plaque that ruptures and precipitates coronary thrombosis. However, as there are no immediate prospects of such prevention, it would be useful to understand the pathogenesis of the later lesions to determine how to intervene and forestall adverse clinical events.

Unfortunately, this is not easy. There is still no neat, linear pathogenetic hypothesis that outlines the sequence of events between the development of the fatty streak and the late lesion, and there may never be one. All the main cell types in a lesion are capable of producing a variety of chemokines, cytokines and growth factors8, 9, 77. All these cells are engaged in a bewilderingly complex torrent of cross-talk. Over time, smooth muscle cells alter their phenotype, replicate, secrete connective tissue matrix proteins, 'imbibe' lipids, undergo apoptosis and die. Macrophages become engorged with lipids, undergo mitosis and die, spilling their lipid into a developing necrotic core. At later stages, endothelial cells are damaged and slough off, creating a nidus for thrombosis. Ultimately, the fibrous cap becomes thin and ruptures, allowing blood access to the tissue factor-rich interior of the lesion—thus precipitating a thrombotic event. These processes are not necessarily sequential; they may transpire concurrently. Moreover, they may not progress linearly with time78, 79. Finally, the complex heterogeneity of late lesions is confounding. In late lesions, some macrophages can be found just beneath the endothelium, and others far below, near the necrotic lipid core. The environments of these two populations of cells are very different, and thus the patterns of gene expression may be very different within a given lesion. Smooth muscle cells alter their phenotype during atherogenesis, and again there may be heterogeneity within the lesion. Consequently, attempts to find commonalities may be impossible without anatomical and functional microdissection. Many unusual suspects have been identified, but their interconnections and 'modus operandi' have yet to be determined. The hope is that from the gang of candidate genes, the ringleaders can be identified and indicted through the use of knockouts, agonists or antagonists, and then intervention strategies can be devised. Gene arrays are being widely used to identify candidate genes, and knockout studies in mouse models offer the cleanest approach to convicting the suspects. Some patterns or common final paths presumably will emerge to bring order to this chaotic situation. Such a common final path function has been postulated for nuclear factor- kappaB (ref. 80), and members of the nuclear receptor family (peroxisome proliferator–activated receptors, estrogen receptors, liver X receptors and retinoid X receptors) may also have such a function77.

Hypercholesterolemia and inflammation: partners in crime
A casual scan of the current literature might give the impression that inflammation has somehow superseded hypercholesterolemia for consideration as the main pathogenic factor in atherosclerosis. Indeed, the main thrust of current research is directed at understanding the complex interactions of cytokines and growth factors in the developing lesion, as discussed above. Rapid advances in the understanding of signaling mechanisms and of regulation of gene expression make this a fertile field of investigation. However, this is not an either–or question. Indeed, a growing body of evidence indicates that oxidized lipids, especially phospholipids but also oxysterols, generated during LDL oxidation or within oxidatively stressed cells, are the triggers for many of the events seen in developing lesions63, 81-84. The structures of these bioactive compounds are similar or identical to those of the oxidized phospholipids in oxLDL that make it a ligand for scavenger receptors (as discussed above). Many represent intact phospholipids in which the polyunsaturated fatty acid at the sn-2 position of the glycerol backbone has been oxidatively degraded but the saturated fatty acid in the sn-1 position remains intact. However, studies strongly indicate that some or all of the biological effects actually reside in the very small percentage of the LDL phospholipids present as the alkyl rather than the acetyl forms; that is the fatty acid in the sn-1 position is ether linked to the glycerol backbone85.

The main point is that oxLDL and its products, including but not limited to the oxidized phospholipids and oxysterols, could well be the elusive initiating factor(s)—the 'injury' to which the artery wall and its component cells respond. The intersections between inflammation in the artery wall and the effects of oxLDL and its products are numerous, as is evident from the partial listing in Table 1. Thus, the lipid hypothesis and the response-to-injury hypothesis not only are compatible but should probably be considered different aspects of a single, shared pathogenetic pathway.

Summary
Atherogenesis is no longer an inevitable consequence of aging—the statin revolution has left no doubt about that. Better control of hypercholesterolemia can undoubtedly be achieved. Many questions remain; however, as will be evident from the reviews in this focus, atherosclerosis research is thriving. The mysteries of the later stages of the disease will yield to the current multidisciplinary attack by academic institutions and by the pharmaceutical industry using the powerful tools of vascular biology and molecular genetics.


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