Current understanding of the mechanisms of atherogenesis

Current Understanding of the Mechanisms Atherogenesis

of

Bruce A. Kottke, MD, PhD

Atherogenesis is a complex process hnrolving several cell types, including endothellal cells, platelets, and smooth muscle cells. l’he development of atherogenesls depends on the modlfrcatlon of the functiin of these cells due to the Interaction of cellular receptors with a variety of peptkle hormones as well as wtth lipoproteln parWes. Approprlate treatment of rlsk factors for atherogenesis depends on this mechanism and must be Indlviduallxed to fit the major mechanisms present In each patient. New tools are emerging to Improve the ability to tallor risk management to fit the needs of pattScular patlent subgroups. (Am J Cardlol1993;72:48C44C)

T

his article provides an overview of the mechanisms of atherogenesis without the confusion introduced by contradictory findings of epidemiology. The process involves primarily the interaction of cells and “particles.” The cells include platelets, endothelial cells, and smooth muscle cells. The particles are lipoproteins. The term “particles” needs to be emphasized. Cholesterol and triglycerides do not occur as such in the blood. The fact that they occur in different types of particles (lipoproteins) is commonly overlooked. Epidemiologists must use great care in the statistical analysis of cholesterol and triglyceride levels. Serious misinterpretations are often made without a thorough understanding of the functions of the particles of which cholesterol and triglycerides are constituents. The forms of cholesterol present in high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) particles are very different physiologic entities with different routes of metabolism and different biologic half-lives. Thus, the analysis of data relating to their sum (i.e., the total plasma cholesterol level) is subject to statistical artifacts. Cells have receptors and enzymes that regulate their interaction with lipoprotein particles. Two major processes occur in atherogenesis and coronary disease. First, there is the slow build-up of a lipid-containing plaque. This is followed by the rapid generation of a clot. Clot formation depends on the balance of the forces that bind platelets to the vessel surface versus the mechanical force of the flow of blood displacing the platelets distally. In unstable angina, this process is continuous and may last for some time before the vessel is obstructed by a clot. ROLE OF ENDOTHEUUM

From

the Mayo Clinic, Rochester, Minnesota. Address for reprints: Bruce A. Kottke, MD, Clinic, P.O. Box 95000, Lakeland, FL 33804.

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IN ATHEROGENESIS

Plaque development depends on 3 processes: endothelial injury, platelet/endothelial interaction, and lipid accumulation. The maior clinically relevant causes of endothelial damage include smoking, antigen/antibody complexes, and homocystine. These effects are counterbalanced by the SEPTEMBER

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effects of several endothelial cell growth factors. At least 3 of these growth factors have been characterized and sequenced and have had their genes cloned. They include acidic fibroblast growth factor.’ platelet-derived endothelial cell growth factor.: and basic fibroblast growth factor.’ Recombinant preparations of these factors are being made by several commercial companies. Once they have been approved for human use, they will undoubtedly be used in clinical practice.l A recent report by Greisler et al5 demonstrated that the incorporation of acidic fibroblast growth factor mixed with fibrin glue into polytetrafluoroethylene (Gore Tex) grafts of an appropriate mesh size allows capillaries to grow between the intersticcs of the graft and to line the graft with the recipient’s own endothelium. This suggests that it is possible and practical to produce long artificial grafts that will be rapidly endothelialized. In another interesting study, Baffour et al6 used a rabbit model in which the arteries to the legs were ligated in stages to produce severe ischemia and the development of “blue toes.” The authors showed that the formation of collateral vessels was increased by the daily intramuscular injection into the thighs of these rabbits of l-3 pg of recombinant basic fibroblast growth factor in an albumincontaining buffer for 2 weeks. This increase in collateral flow was documented by measuring transcutaneous oxygen and by detecting increases in tissue capillaries with the use of microscopy and angiography; in addition, the rabbits’ toes became pink. In the future, clinicians may consider using these potent agents in ischemic cardiomyopathy to stimulate the formation of capillaries. Cofactors arc important in modifying the action of these endothelial cell growth factors.7 Form et

aI8 have shown that when human endothelial cells are grown on plastic in a petri dish, they form a continuous layer. with a cobblestone effect. In contrast, if the plate is coated with laminin or Matrigel (components of basic membranes), the cells form capillaries. ROLE OF PLATELETS IN ATHEROGENESIS Human platelets are unusual cells. They contain a canalicular system that opens up on their surface. This system provides a route for the transport of protein hormones and of other components of their granules to the cell surface. Many active hormones are released by the platelets during aggregation and are secreted into the canaliculi. After aggregation, the platelets take on a “spider” form. All of the hormones released by the platelets have local effects. Local vasospasm probably is not due to a neurogenic mechanism but rather to a local phenomenon resulting from the release of vasoconstrictors from platelet aggregates. The first event following platelet activation is the adhesion of the platelets to endothelial surfaces through the binding of glycoprotein Ia/IIb to endothelial von Willebrand factor or to exposed collagen. Subsequent activation of platelet glycoprotein IIbiIIIa converts this glycoprotein into a fibrinogen receptor. By linking glycoprotein IIbiIIIa receptors on different platelets, fibrinogen becomes the “glue” that aggregates platelets (Figure 1). This binding is orientated to 2 sites on fibrinogen that permit both horizontal and vertical binding of platelets into aggregates.” Both calcium and G proteins are thought to have important roles in this interaction. Recently, investigators have developed a number of peptides that block the glycoprotein IIb/IIIa receptor, thereby preventing platelet aggrega-

FIWRE 1. Diagram of the mechanlsms Involved In platelet adhesion and aggregation. See text for d8talls. Ilb = glycoproteln Ilb; llla = glycoproteln Illa; la = glycoproteln la; lb = glycoproteln ib; VWF = von Wlllebrand factor.

EndotheliumA SYMPOSIUM:

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4%

TULE

I Glycoprotein

lb-llla

Antagonists*

From snake venom Kistrin Bltan Trigramin (3 isoforms) Echistatin From leeches Macrobella decora (Minnesota) Decorsin P/acobel/a omata (Louisiana) Ornatins (6 isoforms) Synthetic SKF 106760 *All of these antagonists recognize the Arg-Gly-Asp (RGD) peptlde.

TABLE II Physical Apolipoproteins Apolipoprotein

Properties

Molecular Mass (kDa)

and Functions

of Human

Distribution

Plasma

Function

A-l

28,000

HDL

Activates

A-II

17,000

HDL

May inhibit HTGL

A-IV

44,500

HDL

B-48

264,000

B-100

550,000

Activates

LCAT LCAT

Obligatory for chylomicron structure LDL, VLDL

Ligand for LDL receptor

C-l

6,600

HDL, VLDL

Activates

LCAT

C-II

8,900

HDL, VLDL

Activates

LPL

C-III

8,800

HDL, VLDL

Modulates hepatlc uptake of Apo Econtaining particles

ROLE OF IJPOPROTEINS

D

33,000

HDL

Cholesterol

E

34,000

HDL, VLDL

Ligand for LDL and LDL receptor-related protein

(a)

300,000-700,000

-

transport

1 Coagulation

HDL = h&h-density lipoprotein; HTGL = hepatic trlglycende lipase; LCAT = lec!thin~holesterol acyltransferase: LPL = lipoprotein lipase; VLDL = very-lowdensity lipoprotein. Reproduced with permission from Lab /nvesf.~~

tion.*@12 The first of these peptides to be developed were isolated from snake venoms or from various leeches. They are listed in Table I. Several synthetic peptides have also been prepared.13 These compounds are called “dysintegrins.” They all recognize the Arg-Gly-Asp binding site on the receptor.14 During the next few years dysintegrins will probably replace aspirin as antiplatelet drugs. They are being cloned, sequenced, and made with recombinant techniques, and they should be available soon. They are effective at picogram concentrations and will be very useful to cardiologists. Platel&derived growth factor: The major reason for blocking platelet aggregation is to prevent the release of platelet-derived growth factor during aggregation. The functions of platelet-derived growth factor include chemotaxis for smooth muscle cells, stimulation of proliferation of smooth muscle cells and of fibroblasts, and stimulation of microsomes to produce elastin, collagen, and muco!%c

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polysaccharides. Platelet-derived growth factor binds to specific receptors, which have been well characterized. Thus, the stimulation of plateletderived growth factor results in the development of the fibrous portion of atherosclerotic plaque. This process is very active in individuals who smoke. The importance of platelet-derived growth factor in intimal proliferation is best demonstrated in the example of late vein graft stenosis following saphenous vein bypass surgery. When this type of surgeq was initiated, the incidence of graft occlusion after 2 years of follow-up was approximately 30%. In a randomized, controlled, double-blind angiographic study, Chesebro et all5 showed that platelet aggregation was inhibited with the use of aspirin and dipyridamole. The incidence of graft occlusion was reduced to 4%. Perhaps the intimal proliferation that occurs after transluminal coronary angioplasty may also be prevented with the use of more effective blockers of platelet aggregation, such as the glycoprotein IIb/IIIa inhibitors discussed above.

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IN ATREROGENESIS

Lipoproteins are composed of lipids that are organized into distinct particles by specific proteins. The proteins of the lipoprotein particles are called apolipoproteins (Table II). In addition to their role in organizing lipids into soluble particles, each apolipoprotein has one or more specific functions. One may think of apolipoproteins as computer codes that direct the protein particles to specific cellular receptors or that direct the metabolism of the particles by regulating cellular enzymes. A complicating feature of this concept is that these apolipoproteins have the ability to transfer from one particle to another and, in the process, change the function or metabolism of the particle. While this results in a complicated system, the system is comprehensible with appropriate chemical definition of the particles. Oversimplifying the measurements that are made of this complex system can lead to serious problems of misinterpretation of functional effects. In the author’s opinion, it is better to address these complexities and gain a more accurate understanding of functional relations. To understand lipid accumulation in the arterial wall, it is necessary to understand the interactions of lipids with endothelial cells, fibroblasts, smooth muscle cells, and macrophages.‘6J7 In fibroblasts and smooth muscle cells, LDL (containing primarily cholesterol esters and apolipoprotein B) binds to LDL receptors. This activates an internalization SEPTEMBER

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process that facilitates the transfer of the LDL receptor and its bound LDL into the cell via the protein clatharin, which is located on a portion of the cell wall called the coated pit. This complex is transported via clatharin-containing endosomes to the lysosomes of the cell. In the lysosome, the proteins are degraded to amino acids, and the cholesterol cstcrs arc hydrolyzed by an acidic cholesterol ester hydrolasc. Although the resulting free cholesterol may potentially be lost from the cell through the ccl1 membrane, most of it is delivered to the microsome, where it is reesterified by acyl-cholesterol-acyl transferase. This recsterification prevents loss through the cell membrane and allows the cell to store cholesterol for use in cell membrane repair. In contrast to smooth muscle cells and fibroblasts, endothelial cells do not have LDL receptors. Instead. they oxidize LDL that is exposed to their surfaces. Their surfaces also contain the enzyme lipoprotein lipase, which is bound to the luminal surface by a heparinoid glycoprotein. This lipase hydrolizes the triglycerides of VLDL and chylomicron particles, converting them to remnants. These remnants arc subsequently removed from the circulation by remnant receptors of the liver. In the arterial wall. the major cell involved in lipid accumulation is the macrophage. Macrophages have receptors for oxidized LDL and remnants but not for LDL. In contrast to the LDL receptors of smooth muscle cells and fibroblasts, thcsc macrophagc receptors are not down-rcgulated by increased concentrations of cellular cholesterol. This results in a massive accumulation of cholesterol esters in macrophages. When these cells become “overstuffed” with cholesterol esters and break up. the released cholesterol esters are in a form that is taken up by adjacent smooth muscle cells. CLINICAL

MANAGEMENT

OF LIPID DISORDERS

The general principles just described are useful in the clinical management of patients with hyperlipoproteinemia. rH There are 3 common types of hyperlipoproteinemia. The first type is characterized by elevated levels of total and LDL cholesterol with normal plasma triglyceride levels. This disease is usually caused by a defect in the LDL receptor, which results in a slowed catabolism of LDL. Occasionally. a similar physiologic effect results from an abnormal apolipoprotein B structure in the region of the molecule that binds to the LDL receptor. In general, the lower the triglyceride

level, the more difficult these patients are to treat. Patients with this type of hyperlipoproteinemia show a minimal response to diet therapy: however, nearly all of these patients can have their lipid levels lowered to normal values with the currently available therapeutic agents and combinations of agents, especially hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitors combined with bile acidsequestering resins. The reason for the striking response to combination therapy is that a promotor region of the hepatic LDL receptor gene is turned off by increased hepatic cholesterol concentrations and is turned on by reduced hepatic cholesterol Icvels. By blocking hepatic cholesterol synthesis with reductase inhibitors and concurrently increasing hepatic cholesterol excretion as bile acids with resins, one achieves a marked reduction of hepatic cholesterol concentrations with a maximal stimulation of the LDL receptor gene. thereby producing receptors that promote LDL removal from the plasma. This effect is further promoted by the fact that most of the reductase inhibitors are preferentially taken up during their first pass through the liver. In a sense, these agents represent a form of gene therapy. The second common type of hyperlipoproteinemia is characterized by increased amounts of VLDL particles due to the overproduction and release of these particles by the liver. This disease is manifested by an elevated plasma triglyceride level accompanied by a cholesterol level that is 20-25% that of the triglyceride level. In some instances this is also accompanied by a low level of HDL cholesterol. These patients respond dramatically to dietary measures. especially even small degrees of weight reduction. In the rare instances where pharmacologic agents are needed, gemfibrozil and niacin arc especially effective. HMG-CoA reductase inhibitors and resins will usually worsen the hyperlipoproteinemia in these patients. The third common type of hyperlipoproteincmia is characterized by elevated levels of both LDL and VLDL. This may be associated with low levels of HDL cholesterol. Patients with this type of hyperlipoproteinemia present with equivalent elevations of cholesterol and triglyceride levels and show an intermediate response to diet therapy. It is difficult to select pharmacologic agents for these patients. The only agent that effectively lowers both LDL and VLDL levels is niacin. Unfortunately, the long-term use of niacin is limited by side effects. The development of a safe hbric acidiHMGCoA reductase inhibitor combination would be very useful for these patients. A SYMPOSIUM:

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A fourth group of patients with lipid disorders consists of those with isolated hypoalphalipoproteinemia. The HDL levels of these patients are not as responsive to therapy as those of patients with low HDL levels associated with hypertriglyceridemia. Gemfibrozil may be effective in mild cases, but in the more severe cases it is necessary to use niacin. Hypoalphalipoproteinemia may also be associated with any of the 3 types of hyperliproteinemia described above. In those cases, it may be necessary to modify the therapeutic approach to address the hypoalphalipoproteinemia as well as the other lipid disorders. The three common types of hyperlipoproteinemia as well as hypoalphaliproteinemia all increase the risk of atherosclerotic disease. Evidence from the Helsinki Heart Study’” and from other studies indicates that HDL particles are at least as important as LDL and VLDL particles as risk factors for atherosclerotic disease. PREVENTIVE CARDIOLOGW NEEDS AND FUTURE DIRECTIONS Clinicians currently have available many of the tools needed to modify significantly the risk factors for atherogenesis. In addition, a few new tools are evolving. Although assays for the apolipoproteins have been available for some time, they have yet to be standardized. Many of the commercial assays depend on methods that are affected by variations in the size of the particles, such as nephelometry, radioimmunodiffusion, and electroimmunoassay. When the same protein is associated with particles of more than one size (which is usually the case in hyperlipoproteinemia), such assays will not provide consistent results. In addition, many assays are done using serum. These are also inappropriate, since the cellular proteases released during the preparation of serum can degrade the apolipoproteins, resulting in artificially low levels. Additional problems are evident when one looks at commercially available assays for lipoprotein(a). These assays measure lipoprotein(a) in mg/dL units. Since apolipoprotein(a) can vary widely in size (350-800 kDa in molecular mass) on the basis of at least 24 alleles,“‘changes in measured concentrations in mg/dL units may reflect either an alteration in the size of the particle or a change in the molar concentration of the particles. To reflect the true state of affairs, particles with a variable molecular weight, such as lipoprotein(a), should be measured in mmoliliter units. Attempts to develop such assays are currently underway. Clearly, to understand the complex physiology 52C

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and cell biology of lipoprotein metabolism, we need to develop tools to quantify the specific subparticles of lipoproteins that circulate in the plasma. The current attempts to separate HDL into particles containing only apolipoprotein A-I (lipoprotein A-I) from those containing both apolipoprotein A-I and apolipoprotein A-II (lipoprotein A-I/A-II) is an important step in that direction. Another risk factor for atherogenesis is homocysteine.” It is well known that homocysteine is toxic to endothelium. In addition to heterozygotes with the genetic disease homocysteinemia, any folic acid deficiency such as that induced by antibiotics (e.g., trimethoprimisulfamethoxazole) can elevate homocysteine levels. Recent reports suggest that elevated levels of homocysteine occur in 15% of patients with angiographically detected coronary artery disease.” Since this disease can be treated effectively with folic acid, an agent with low toxicity, it is important that this risk factor be identified and treated. Finally, it would be extremely helpful if one could accurately detect asymptomatic coronary artery disease manifested by coronary artery lesions that are not hemodynamically significant and, therefore, not identified by the usual stress testing. We know that patients with 30-40% stenotic lesions often present with acute myocardial infarction or experience sudden death when a thrombosis develops in addition to these “nonobstructive” plaques. Currently, the author and others are investigating the potential usefulness of ultrafast computed tomography imaging of coronary calcification as a tool for addressing the problem of hemodynamitally insignificant lesions of the coronary artery. If such patients can be identified, they would be an ideal targeted group for aggressive intervention for the control of risk factors. Early work by Simons et aP indicates that if no coronary calcification is detected by this technique, there is a 98% chance that the patient does not have hemodynamically significant coronary artery disease. On the other hand, if even small amounts of calcification are detected, there is a 90% chance that the patient has at least 10% narrowing of r 1 coronary arteries. The latter patients can be used as a target group for the aggressive control of the specific risk factors present in the individual patient. We are currently conducting studies using this technique in a large, normal, asymptomatic population to define further its usefulness as a screening tool. Because a patient can be scanned in I 10 minutes, SEPTEMBER

9. 1993

DISCUSSION

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t1> 1w\ernent

mcmtTme

ccmfx’“‘“t\.

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coronarv

I O(tl 1

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3.3. S,,,n,,,en I. ,\h f f< I-. I’totrt~w~cr KS. Kuratil 1, L<~ltus .I(‘. Kumcki I J. ‘The ,Arg-( ity-/2\p ( RCiI)) rcc~ylnltx,n \I,C r,t pl;1tclct plyctlproteln llt+llla on II~I~,I~T~.w~ ptatclct\ I\ accc\\itdc 113h@tllmity mncromdcculc~ Hl~xnl 15. (‘hed,r,, ,111. (‘leincnt\ If’. Fustrr &T I I\: KI I If:: .Jr. \‘IIc\,I
I’. t Ihehach 1 K. Smith HC. Uardstq Kf.. f’luth .I K. \Vdl;rte f
heart

acid deficiencies.

14”: of patients with disease have elevated levels of

‘T‘rimethoprim/sulfamcthoxa-

Dr. Kottke: The best way to diagnose it at the present time is debatable. Most authorities would recommend a dietary methionine load. which can bc done for approximately 6 hours. to obtain a homocysteine level. The current measures of homocvstcine involve amino acid analvsis with current tigh-performance liquid chr&atography techniques. That is not difficult to do. We must stop A SYMPOSIUM

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ISCHEhllA

53c

thinking that atherosclerosis is a problem solely of diet or of any one thing. Atherosclerosis takes many different forms, and each form must be treated differently. We must individualize the treatment to fit the patient. Dr. Elliott M. Antman (Boston, Massachusetts): Dr. Kottke, I would like your comments on the use of compounds such as the glycoprotein IIb/IIIa receptor inhibitors. There must be monoclonal antibodies that are available. Although these may increase our ability to block the receptor, I am concerned that we may be putting patients at an increased risk for bleeding. I am also concerned about the increased costs that this therapy would incur in treating patients with coronary heart disease and a variety of other diseases as well. Dr. Kottke: Although bleeding is an important concern, I am not aware of an excessive number of bleeding disorders associated with the use of these antibodies. You have to understand that they can be used only once in a given situation, and that the degree of glycoprotein IIb/IIIa inhibition cannot

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be controlled precisely because the binding of the antibodies is going to be variable. In regard to inhibitors of glycoprotein IIb/IIIa that are not antibodies, the good news is that there are 10-15 compounds available. It is our hope that 1 or 2 of them will prove useful. In regard to cost, these inhibitors would have their first use in unstable angina, where platelet aggregation is the primary problem. Glycoprotein IIb/IIIa is the final common pathway. Until now, everything we have used, such as thromboxane, thrombin, and plateletactivating factor, has dealt with inputs into that pathway. Now we are treating patients at the receptor level. This necessitates learning the right dose of inhibitors to use. It certainly will necessitate trying the various compounds to see which ones are effective. My point is, we have a lot of potential tools to try. I believe that blocking that receptor may be an extremely powerful technique. If it is used in unstable angina, it is going to be a lot less expensive than other treatments currently in use, and it may not have to be used long term.

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