Atherosclerosis: The low-density lipoprotein receptor hypothesis

Atherosclerosis: The low-density lipoprotein receptor hypothesis

PROGRESS IN ENDOCRINOLOGY AND METABOLISM Atherosclerosis: The Low-Density Receptor Hypothesis Joseph Recent biochemical, structural studies hep...

2MB Sizes 2 Downloads 63 Views

PROGRESS

IN ENDOCRINOLOGY

AND METABOLISM

Atherosclerosis: The Low-Density Receptor Hypothesis Joseph Recent

biochemical,

structural

studies

hepatic

human

genetic,

have cells,

lymphocytes,

and

cells, utilize

a specific

the

low-density

way,

The critical surface

component binds

plasma.

is

of

of its

is

on the

cell

the

major

uptake

the

for

receptorwith

use

When

in

form

paper

the

normal

cholesterol We this

conclusion

of LDL is

when

lend

“normal” in Western

the

LDL

by

explain

of

atherosclerosis

The

on

derived

LDL-

the

support

studies-namely, level

the

plasma

data

previously

con-

at low

a mechanism

biochemical

plasma

nonwith

might

“normal”

levels.

that

allow

is maintained

occurrence with

cholesterol

demiologic

is to

discuss

In

high-affinity

themselves

hypothesis

humans

re-

hyper-

then

a

ensues.

the

supply

to

and

the hypothesis

receptor

to

widespread

plasma,

of

at a time

levels.

the

present

in plasma

which

accumulates

the

function LDL

centration

pathway

in

atherosclerosis

we

cells

in

familial

of

cell-surface

LDL

as

the degradation

re-

lipoprotein

levels

this

in

cellular

the

absent, of

chohuman

intralysosomal

lipoprotein

form

cholesterolemia,

of by

genetically

homozygous

of

the

high

hepatic

pathway

synthesis.

is

path-

cholesterol.

of this

cholesterol

membrane ceptor

(LDL) with

LDL,

the

muscle pathway,

S. Brown

impaired, very severe

receptor

followed

degradation lease

smooth

lipoprotein

Cellular LDL

non-

fibroblasts,

metabolic

themselves

and Michael

ultra-

that

as

aortic

lesterol-carrying bound

such

high-affinity that

and

disclosed

lipoprotein

to supply

a specific

L. Goldstein

Lipoprotein

LDL

to the

from

epi-

that

the

of LDL-cholesterol

man is unphysiologically

high.

T

HE MOST STRIKING biochemical abnormality in human atherosclerosis is the accumulation of massive amounts of cholesteryl esters in the core of the atheromatous plaque.‘,2 The oldest and simplest explanation for the deposition of cholesteryl esters in the artery wall is the insudative theory first postulated by Virchow more than 100 yr ago. In its modern form, this theory states that the primary lesion in atherosclerosis involves an insult to the arterial endothelium.3”4 While the cause of the initial endothelial damage may vary from patient to patient and fr’om site to site, the end result is remarkably constant. As the normal barrier function of the endothelium breaks down, plasma constituents, including cholesterol-carrying lipoproteins, penetrate into the inner layer (the intima) of the artery wall. As a result of this injury, smooth muscle cells in the middle layer (the media) proliferate, perhaps in response to a growth factor relased from platelets that aggregate over areas of endothelial damage.’ The proliferating smooth muscle cells then invade the intima. where they ingest, degrade, and excrete the foreign material. However, the cholesterol that is carried into the artery wall by the plasma lipoproteins is so insoluble that it can be removed by the smooth muscle cells only at a relatively slow rate and its pro-

From

the Division

Healrh Science Receivedfor Supported

of Medical

Center,

publication

Genetics,

of Internal

Medicine,

University

of Texas

18. 1977.

the National

Institutes

of Health,

the National

Foundation-March

of

Heart Association.

requests should be addressed

University of Texas

Departmenr

Texas.

March

b_v grants from

Dimes. and the .4 merican Reprint

Dallas.

Health Science

‘CJ1977 bv Grune & Stratton,

to Dr. Joseph

Center,

Inc. ISSN

L. Goldstein,

5323 Harry

Department

Hines Blvd., Dallas,

of Internal

Texas

Medicine,

75235.

0026-0495.

Metabolism, Vol. 26, No. 11 (November), 1977

1257

1258

GOLDSTEIN

AND BROWN

gressive accumulation leads to the relentless growth of the atheromatous plaque.3-5 A simple way for the body to limit the penetration of cholesterol into the artery wall would be to eliminate cholesterol from plasma, In beginning to unravel the complex pathogenesis of atherosclerosis, one can first ask the question: Why is cholesterol present in plasma‘? During the past 4 yr, our laboratory, as well as those of other investigators, have begun to provide answers to this question through studies of the interaction of plasma lipoproteins with human cells. These studies have disclosed that extrahepatic cells require cholesterol for plasma membrane synthesis and that certain lipoproteins function to fulfill this requirement by transporting cholesterol through the plasma from its site of origin in the liver or intestine to its site of utilization by extrahepatic cells. In carrying out this cholesterol transport, the body is faced with a dual problem. On the one hand, it must maintain plasma lipoprotein concentrations high enough to supply its cells with cholesterol, and on the other hand, it must keep plasma cholesterol levels low enough to prevent insudation into the artery wall with resultant atherosclerosis. This dual problem is solved by the existence of a high affinity-surface receptor on body cells that allows the cells to bind and take up cholesterol from lipoproteins at a time when the lipoproteins are present at low concentrations in plasma and interstitial fluid. Such a high-a~nity lipoprotein receptor has been demonstrated on the membranes of a variety of human cells, including fibroblasts, lymphocytes, and arterial smooth muscle cells. The receptor specifically binds the most abundant cholesterol-carrying lipoprotein in human plasma, low-density lipoprotein (LDL).5” Binding of LDL to the high-a~nity LDL receptor constitutes the first step in a pathway by which cells take up the lipoprotein and use its cholesterol. Human subjects exhibit a variety of genetic defects in this LDL receptor pathway that produce discrete blocks in the binding, uptake, or degradation of LDL by cells and thereby cause a marked elevation of LDL in the plasma followed by premature atherosclerosis.5-8 In this article, we first review the biochemical, genetic, and ultrastructural evidence that has led to the delineation of the receptor-mediated LDL pathway in human cells. We then discuss the role that this LDL pathway may play in maintaining cholesterol homeostasis in the human body. Finally, we present a hypothesis to explain how the LDL pathway may act to prevent the accumulation of cholesteryl esters and thus protect against atherosclerosis. Two Forms of Cholesterol: Free and Ester$ed Cholesterol exists in the body in two forms, namely, as free cholesterol and as cholesteryi esters. Free cholesterol is the functionally important form of the sterol. It is a required structural component of the plasma membrane of all mammalian cells. Free cholesterol also serves as a precursor for the synthesis of steroid hormones and bile acids. For purposes of storage and transport. cholesterol is attached to long-chain fatty acids to form cholesteryl esters. At body temperature, cholesteryl esters form liquid crystals that can be stored in tissues more easily than can solid crystals of free cholesterol. About threefourths of the cholesterol that circulates in plasma LDL is in the form of cholesteryl ester.9.‘a

1259

ATHEROSCLEROSIS

% BY

COMPONENT Apoprotein

Fig. 1. Schematic diagram showing the structure and composition of plasma LDL. Plasma LDLis depicted OS a lipoprotein particle composed of an apolar core of esterified cholesterol that is surrounded by a polar coat composed of phospholipid, free cholesterol, and apoprotein B.

L

WEIGHT

B ~25%

Tot01 i~p,ds -75% Cholesterol -Flee -Ester,f,ed ~ Phosphol,p,ds-------25% Glycertdes ~

7% 35% 8%

Figure 1 shows in diagramatic fashion the structure of LDL. The bulk of this lipoprotein consists of a core of neutral lipid, composed mostly of cholesteryl ester. This core is surrounded by a polar coat consisting of free cholesterol, phospholipid, and a protein called apoprotein B.9 Studies in tissue culture have demonstrated that in order for cells to use the cholesteryl esters contained within the core of LDL, they must take up the lipoprotein intact.5~7 LDL PATHWAY

The pathway by which human cells gain access to the cholesteryl esters consteps in this tained in LDL has been called the LDL pathway. 5~7The sequential pathway as delineated in human fibroblasts are illustrated in Fig. 2. When cells are in need of cholesterol, they synthesize a specific receptor that becomes localized to the cell surface.” LDL binds to this receptor with high affinity, and

LDL BINDING

-C%-S

-

LYSOSOMAL REGULATION OF HYDROLYSIS-MICROSOMAL ENZYMES

Fig. 2. Sequential steps in the LDL pathway in cultured human fibroblosts. The numbers indicate the sites at which mutations have been identified: (1) abetalipoproteinemia; (2) familial hypercholesterolemia, receptor-negative; (3) familial hypercholesterolemio, receptor-defective; (4) familial hypercholesterolemia, internalization defect; (5) Wolman syndrome; and (6) cholesteryl ester storage disease. HMG CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase; ACAT, acyl-coenzyme A:cholesterol acyltransferase. (Modified from Brown and Goldstein6 and reproduced with permission of the publisher).

GOLDSTEIN

1260

AND

BROWN

the bound LDL is then taken up by the cell through a process resembling adsorptive endocytosis.‘2m1s During this uptake process, the plasma membrane to which the LDL is bound invaginates into the cell to form an endocytic vesicle. The endocytic vesicle then migrates through the cytoplasm until it reaches a lysosome, whereupon the membranes of the endocytic vesicle and the lysosome fuse, exposing the bound lipoprotein to a variety of acid hydrolytic enzymes.13*‘6-‘* The protein of LDL is hydrolyzed by lysosomal proteases to amino acids.r3.rh The cholesteryl esters of LDL are hydrolyzed by a lysosomal acid lipase.‘7.‘8 In contrast to the cholesteryl esters, which are too nonpolar to cross cellular membranes, the free cholesterol that is produced is able to passively cross the lysosomal membrane and gain access to the cellular compartment, where it is used for membrane synthesis.‘8-20 Having obtained cholesterol from LDL, the cell suppresses its own cholesterol synthesis by reducing the activity of the rate-controlling microsomal enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase).2i If any excess cholesterol enters the cell through the LDL pathway, it is stored by the cell in the form of cholesteryl ester droplets. To trigger the formation of these cholesteryl esters, the excess free cholesterol activates acylCoA:cholesterol acyltransferase (ACAT), a microsomal enzyme that attaches a long-chain fatty acid to free cholesterol to form cholesteryl esters.2?%23Thus, the acquisition of cholesterol from LDL causes a suppression of one microsomal enzyme, HMG CoA reductase, and a reciprocal activation of another microsomal enzyme, ACAT.” DEFECTIVE

LDL RECEPTOR

IN FAMILIAL

HYPERCHOLESTEROLEMIA

The existence of the LDL pathway was first postulated on the basis of biochemical studies of human fibroblasts grown in tissue culture. The interpretation of these studies was aided greatly by the existence of a variety of human mutations. That is, fibroblasts were available from patients with genetic diseases that produce discrete blocks at specific sites in this pathway. To date, six such mutations have been identified, five affecting the LDL pathway at the cellular level and one affecting the secretion of plasma LDL itself (see legend to Fig. 2). The mutation that has proved to be most informative is the one that produces familial hypercholesterolemia, an autosomal dominant disease that produces both hypercholesterolemia and premature atherosclerosis.i4-26 In patients who have inherited a single copy of the gene for familial hypercholesterolemia (i.e., heterozygotes), the total plasma cholesterol level is about 300-500 mg/dl from birth, but symptoms do not develop until the third to the sixth decade, when tendon xanthomas and coronary heart disease appear. About I in 500 persons in the general population and about 1 in 20 patients with myocardial infarction have the heterozygous form of familial hypercholesterolemia-one of the commonest simply inherited disorders in man.27 Rare patients inherit two copies of the familial hypercholesterolemia gene (i.e., homozygotes), and in them the clinical picture is much more severe. The total plasma cholesterol level usually exceeds 800 mg/dl; cholesterol accumulates in widespread sites, producing cutaneous planar and tendon xanthomas during the first few years of life. Signs of coronary heart disease are nearly always evident before age 20 yr.24-26

1261

ATHEROSCLEROSIS

Cultured fibroblasts from patients with homozygous familial hypercholesterolemia have been shown to possess three types of genetic defects in the LDL receptor. Cells from one type of homozygote are completely unable to As a result of this primary lack of LDL recepbind LDL at the cell surface. 8.11m14 tors, these homozygote cells show the expected secondary abnormalities in cellular cholesterol metabolism, including (1) a marked reduction in the rate of (2) a marked decrease in the rates at which the cellular uptake of LDL; 12m14,28 cells hydrolyze both the protein and cholesteryl ester components of LDL:13.” (3) an inability of LDL to elevate the cellular content of free and esterified cholesterol;” (4) a failure of LDL to suppress the activity of HMG CoA reductase and hence cholesterol synthesis;2’,29,30 and (5) a failure of LDL to stimulate the synthesis of cholesteryl esters.?* To date, detailed analyses have been performed on fibroblast strains derived from 22 patients with the classic clinical picture of homozygous familial hypercholesterolemia. The cells from 12 of these patients have shown a complete lack of LDL receptors, as described above. These patients have been called receptornegative homozygotes.6,31 Nine of the patients have shown a different type of mutation in the receptor such that about 57;- 20”; of normal binding activity is expressed. These patients are designated as receptor-defective homozygotes.8,3’ In addition to these two classes, we have recently identified one patient whose fibroblasts are unique. Cells from this strain are able to bind normal amounts of LDL at the receptor site but are unable to internalize the LDL after it is bound to the receptor.32 The existence of this mutation indicates that the adsorptive endocytosis of LDL by cells requires at least two functionally distinct active sites, each of which can be altered by mutation: namely, an active site that is required to bind LDL, and an active site that mediates the internalization of receptor-bound LDL. Recent genetic evidence suggests that both active sites are present on the same receptor molecule. ULTRASTRUCTURAL

IDENTIFICATION

OF THE

LDL RECEPTOR

All of the above conclusions regarding the LDL pathway were based on biochemical studies of the metabolism of lZ51-labeled LDL. To confirm that LDL was indeed being taken up by endocytosis and that specific membrane receptors were involved, Anderson and co-workers recently turned to the use of the electron microscope. 11.33Using ferritin-labeled LDL as a probe for electron microscopic studies, these investigators showed that in fibroblasts about 707, of the LDL receptor sites are concentrated in short (0.5 pm) segments of plasma membrane where the membrane appears indented and coated on both of its sides by a fuzzy material. “,33 These so-called “coated regions,” which constitute less than 2”, of the total surface membrane of fibroblasts, have been observed previously in a variety of other cell types, where they have been postulated to play a role in the specific uptake of proteins by adsorptive endocytosis.34m36 When the LDL-ferritin complex is first allowed to bind to the coated regions of normal fibroblasts at 4°C and the cells are then warmed to 37”C, the coated regions can be observed to invaginate to form coated endocytic vesicles. Within 10 min most of the surface-bound LDL-ferritin has been internalized within such coated vesicles, which then migrate through the cytoplasm and ultimately fuse with lysosomes.37

GOLDSTEIN

1262

AND

BROWN

In similar studies performed with fibroblasts from receptor-negative familial hypercholesterolemia homozygotes, a normal number of coated regions was seen on the plasma membrane but no LDL-ferritin was observed to bind to these regions and none entered the cell. ‘1~‘3.37Thus, the electron microscopy studies confirmed the original conclusions of the biochemical studies indicating that high-affinity LDL receptors exist on the surface of normal fibroblasts and that these LDL receptors are absent in these mutant cells. REGULATION

OF THE

LDL RECEPTOR

As discussed above, the LDL pathway functions to supply cells with the free cholesterol that they require for plasma membrane synthesis. If any excess cholesterol enters the cell through the pathway, it is stored by the cell in the form of cholesteryl oleate and other cholesteryl esters. However, if LDL uptake always occurred at maximal rates cholesteryl esters would rapidly accumulate. Under these conditions, cells would become stuffed with cholesteryl esters to the point of cellular toxicity. Recent studies have disclosed that the major way in which human fibroblasts and vascular smooth muscle cells protect themselves against such cholesterol overaccumulation is by exerting a tight control 38139 over the number of LDL receptors. Thus, when cells are deprived of cholesterol, they synthesize a large number of LDL receptors and acquire the ability to take up LDL at a very high rate. On the other hand, when the intracellular level of free cholesterol becomes restored and cholesteryl esters begin to accumulate in the cell, the synthesis of LDL receptors becomes suppressed and the rate of LDL uptake declines. This turning off of LDL receptors prevents the cell from overaccumulating cholesterol through the LDL pathway.38,39 The data in Fig. 3 illustrate the importance of the regulation of the LDL

0

24 HOURS

48 OF EXPOSURE

72

96

TO LDL

Fig. 3. Accumulation of cholesteryl estersand suppression of ‘251-LDLbinding activity in normal human fibroblasts incubated with LDL. Monalayers of cells were grown for 3 days in medium containing 10% lipoprotein-deficient serum. 32 On day 4 of cell growth (zero time), each dish of nonconfluent cells received 2 ml of growth medium containing 10 pg protein/ml of unlabeled LDL. Every 24 hr throughout the experiment, the medium in each dish was replaced with fresh medium containing the same amount of unlabeled LDL. Each day, as indicated, the medium from one set of duplicate dishes was removed and replaced with 2 ml of medium containing 25 pg protein/ml of 12sl-LDL (80 cpm/ng). After incubation at 37°C for 2 hr, the cells were harvested for measurement of the amount of 125~-~~~specifically bound to the cell surface receptor (ban).14 At the same time, a second set of duplicate dishes was washed, harvested, and pooled (two dishes/ sample) and the cellular content of cholesteryl esters was determined by gas-liquid chromotography (0). l9

1263

ATHEROSCLEROSIS

receptor in preventing an overaccumulation of cellular cholesteryl esters. In this experiment, fibroblasts were induced to synthesize a maximal number of LDL receptors by prior growth in the absence of cholesterol-carrying lipoproteins. Following this preliminary growth period (i.e., at zero time), LDL was added to the culture medium at a concentration of 10 pg protein/ml. Because of the large number of LDL receptors, the uptake of LDL produced a dramatic rise in the cellular content of cholesteryl esters within the first 24 hr. However, this rise itself produced an equally dramatic fall in the number of LDL receptors per cell. Therefore, on subsequent days, even though the same amount of LDL was maintained in the culture medium, the cellular content of cholesteryl esters actually decreased because the cells were no longer able to take up and degrade large amounts of LDL. If the LDL receptor activity were not subject to this type of feedback regulation and the cells had continued to take up LDL at the initial rate, the accumulation of cellular cholesteryl esters would have been enormous. This ability to regulate precisely the number of LDL receptors appears to be the factor that prevents normal parenchymal cells that are exposed continuously to LDL from turning into foam cells of the type seen in atherosclerotic lesions.39 OVERACCUMULATION OF CHOLESTERYL ESTERS RESULTING FROM

UNREGULATED

LDL

UPTAKE

Because such a sophisticated control mechanism exists to protect cells from overaccumulating LDL-cholesterol in vitro, it has been difficult to study experimentally the pathologic consequences of an overaccumulation of cholesteryl esters in cells. Recently, Basu and co-workers have devised a method to induce cells to take up LDL by a mechanism that bypasses the LDL receptor.‘“.4’ These workers took advantage of the fact that the surface of all mammalian cells contains a variety of negatively charged groups (mostly sialic acid residues). Since LDL normally has a net negative charge, it does not bind to these anionic sites. They reasoned, however, that if the charge on LDL were converted from negative to positive, then the lipoprotein might bind to the various anionic sites on the cell surface and enter the cells by a mechanism that did not involve the LDL receptor. To give LDL a positive charge, the lipoprotein was coupled to a tertiary amine, N,N-dimethyl-1,3-propanediamine (DMPA). which converted the negatively charged amino acids of LDL into positively charged amine groups.40 It was possible to show both biochemically and ultrastructurally that this modified polycationic DMPA-LDL binds in large amounts to the cell surface and is subsequently taken up by endocytosis and delivered to lysosomes, where its protein and cholesteryl ester components are hydrolyzed.40,4’ The conclusion that this polycationic LDL enters fibroblasts by a mechanism that bypasses the LDL receptor was supported by the finding that rapid binding, uptake, and degradation of the DMPA-LDL occurred in the homozygous familial hypercholesterolemia cells that lack functional LDL receptors.40,4’ Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL

1264

GOLDSTEIN

AND BROWN

receptor. As a result, unlike the case with native LDL, the polycationic DMPA-LDL produced a massive increase in the content of both free and esterified cholesterol within the cell. After several days of incubation with 10 pg protein/ml of DMPA-LDL, fibroblasts and vascular smooth muscle cells contained approximately 50-fold higher levels of cholesteryl esters than did cells exposed to an equal concentration of native LDL.39,41 The accumulation of this massive amount of cholesteryl esters in these cells gave rise to a biochemical picture similar to that seen in smooth muscle cells of the artery wall that accumulate massive amounts of cholesteryl ester during the process of atherosclerosis.39 When exposed to polycationic DMPA-LDL, fibroblasts and aortic smooth muscle cells accumulated so much cholesteryl ester that large numbers of neutral lipid droplets were visible at the light microscopic level using Oil Red 0 staining. 39,4’Moreover, by electron microscopy, these intracellular lipid droplets were observed to lack a typical unit membrane and to be clustered in regions of the cell that were rich in rough endoplasmic reticulum.39s4’ None of these features were prominent in fibroblasts or smooth muscle cells incubated with native LDL, even when the lipoprotein was present at high concentrations. These data indicate that when cells are forced to take up LDL through a mechanism that bypasses the normal regulatory mechanism, they begin to resemble the type of foam cells seen in the artery wall during atherosclerosis.39 The factors within the artery wall that trigger the unregulated uptake of native LDL in vivo are not yet known. However, the ability to reproduce the pathologic features of atherosclerosis in a simple in vitro system should now make it possible to identify and characterize at the molecular level the factors that reverse as well as cause the type of abnormal cholesterol ester deposition that occurs in the cells of the artery wall during the atherosclerotic process. OVERALL REGULATION OF LDL AND CHOLESTEROL METABOLISM IN CULTURED FIBROBLASTS

Figure 4 summarizes the integrated pattern of regulation of LDL and cholesterol metabolism as it has been elucidated in cultured human fibroblasts.5-7 When fibroblasts are grown in the presence of normal serum containing LDL, they establish a steady state in which HMG CoA reductase activity (and hence cholesterol synthesis) is low and the cells derive the small amounts of cholesterol that they need by means of a small number of LDL receptors. Under these conditions, the activity of ACAT is held at an intermediate level so that the rate of synthesis of cholesteryl esters equals their rate of hydrolysis (Fig. 4, “steady state with LDL present”). The delicate balance inherent in this regulated steady state is disclosed only when LDL is removed from the culture Under these conditions, the number of medium (Fig. 4, “no LDL present”). LDL receptors and the activity of HMG CoA reductase greatly increase, while cholesterol esterifying activity declines. Since LDL is absent from the culture medium, the LDL receptors are not able to supply the cell with cholesterol and hence the cholesterol required for membrane formation is derived both from accelerated de novo synthesis and from a net hydrolysis of cholesteryl esters stored within the cell. When LDL is added back to the culture medium (Fig. 4, “initial response to LDL”), the lipoprotein is bound at the receptor site, internalized, and degraded to yield free cholesterol. The liberated sterol, in turn,

1265

ATHEROSCLEROSIS

NO LDL P#?ES..NJ

NVIJIAL RESPONSE

-LDL i

TO L DL

tLbL :

\

\

STEADY

SJAJE

WITH LDL PRESENT

I Z

i

.’

Fig. 4. Cyclic changes in cholesterol metabolism that occur in cultured human ftbroblasts when LDL is removed from the culture medium (- LDL) and is subsequently returned to the medium (+LDL). The relative level of each constituent is indicated by the size of the square. HMG CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase; ACAT, acyl-coenzyme A:cholesterol acyltransferase. (From Brown and Goldstein,6 reproduced with permission of the publisher).

suppresses de nova cholesterol synthesis and stimulates the esterifying system so that excess cholesterol can be stored as cholesteryl esters. When sufficient cellular cholesterol has accumulated, the number of LDL receptors becomes suppressed and the cells return to their original steady state (Fig. 4, “steady state with LDL present”), thus completing a metabolic cycle. This “steady state with LDL present” duplicates the condition of most cells in the body. ROLE OF THE

LDL RECEPTOR

IN VIVO

All of the aspects of the LDL pathway that have been described above for cultured fibroblasts have also been documented in cultured human arterial smooth muscle cells39.42,43and in cultured human lymphoblasts44~45 as well as in cultured cells from a variety of animals such as mouse L cells and Chinese hamster ovary cells.’ To determine whether the LDL pathway could be demonstrated in human cells directly upon their removal from the body, studies have also been performed on freshly isolated lymphocytes.46,47 Since lymphocytes. like other blood cells, are continuously exposed to LDL, it would be expected that lymphocytes taken directly from the bloodstream should synthesize cholesterol at low rates and express only a small fraction of their maximal number of LDL receptors. The fibroblast studies also predicted that if these lymphocytes were incubated for an appropriate time in serum devoid of LDL. both the number of LDL receptors and the ability to synthesize cholesterol would increase. The data in Fig. 5 show that this is precisely what was observed. The bars represent LDL receptor activity as assessed either by the ability of the cells to bind ‘25I-LDL at the receptor site at 4°C or by the ability of the cells to degrade

GOLDSTEIN

I

AND

BROWN

BINDING 400

DEGRADATION

fz ;;1 9

300

_

2 J &k T:! 200 lo JR LP z ‘00 2 s 0

24 TIME

IN CULTURE

48

72

0

(hours)

Fig. 5. Increase in LDL receptor activity in lymphocytes during incubation in the absence of lipoproteins. Lymphocytes were isolated from the venous blood of healthy individuals and the cells were incubated at 37°C in medium containing 10% human lipoprotein-deficient serum.46 After the indicated interval, LDL receptor activity was assessed by measurements of the highaffinity binding of 1251-LDl at 4°C (solid bars) and the high-affinity degradation of 1251-LDL at 37°C (hatched bars) as previously described.46 normal

lZ51-LDL (which requires that the LDL first bind to the receptor). When fresh lymphocytes are initially isolated from normal subjects, they have a relatively small, but detectable ability to bind and degrade ‘251-LDL. However, when these cells are incubated in the absence of lipoproteins, they induce the synthesis of LDL receptors and thus acquire a progressive increase in their ability to bind and degrade ‘251-LDL.46 To determine whether the LDL receptors that appear on fresh lymphocytes are the same genetically as the LDL receptors in cultured fibroblasts, lymphocytes from patients with homozygous familial hypercholesterolemia were studied. Figure 6 shows the results of measurements of LDL binding and degradation after incubation for 72 hr in the absence of lipoproteins. In contrast to the cells from normal subjects, lymphocytes from patients with homozygous familial hypercholesterolemia developed less than 2% of normal LDL receptor activity either as measured by binding or degradation of ‘2sI-LDL.46 Ho et al. have also recently shown that normal human lymphocytes isolated freshly from the body exhibit a very low rate of cholesterol synthesis, presumably because they have just been removed from a medium in which they have had abundant access to LDL.4’ However, when these cells were incubated in the absence of lipoproteins, cholesterol synthesis increased markedly and this increase was prevented when LDL was included in the incubation medium. Moreover, when the lymphocytes were first allowed to develop enhanced cholesterol synthesis by incubation in the absence of LDL for 72 hr and then LDL was

Fig. 6. LDL receptor activity in lymphocytes from normal subjects (e) and patients with homozygous familial hypercholesterolemia (A). lymphocytes were isolated from the venous blood and the cells were incubated for 72 hr at 37°C in medium containing 10% human lipoprotein-deficient serum.46 LDL receptor activity was assessed by measurements of the high-affinity binding of 1251-LDLat 4’C (A) and the high-affinity degradation of ‘2s~LDL at 37°C (6) as previously described.46

25

5 0

1267

ATHEROSCLEROSIS

added back to the system, prompt suppression of cholesterol synthesis was observed. Thus, when deprived of LDL, lymphocytes, like fibroblasts, develop both an enhanced number of LDL receptors and an enhanced ability to synthesize cholesterol. When LDL is once again made available, the cells preferentially utilize the LDL-cholesterol and suppress cholesterol synthesis. That this suppression of cholesterol synthesis by LDL requires the LDL receptor is indicated by the finding that when lymphocytes from patients with homozygous familial hypercholesterolemia were allowed to develop high rates of cholesterol synthesis and then were exposed to increasing amounts of LDL, the mutant cells showed a complete resistance to suppression of cholesterol synthesis by LDL.47 Thus, LDL metabolism in fresh human lymphocytes is similar to that in cultured fibroblasts by both biochemical and genetic criteria. IMPLICATIONS OF THE LDL PATHWAY NORMAL HUMAN PHYSIOLOGY

FOR

The extensive information that has emerged from the study of lipoprotein metabolism in isolated cells has raised two important questions regarding LDL metabolism in the whole organism: First, what role does this LDL receptor pathway play in the body? Second, how does the deficiency in LDL receptors in familial hypercholesterolemia homozygotes lead to a massive overaccumulation of LDL in the plasma and produce accelerated atherosclerosis? Figure 7 shows a scheme that we propose as a working model for the metabolism of cholesterol-carrying lipoproteins in the body. This scheme integrates the knowledge gained from studies in isolated human cells with the critical information that has been gathered by other investigators, such as Havel, Glomset, Frederickson, Levy. Steinberg, Dietschy, Zilversmit, Bilheimer, Myant, and others who have studied the metabolism of cholesterol-carrying lipoproteins in experimental animals and in man.48 It is clear that the major site for control of plasma cholesterol transport resides in the liver. The liver receives dietary cholesterol in the form of remnants derived from the metabolism of chylomicrons. The liver in turn excretes cholesterol into the bile either as neutral sterol or as bile acids, the breakdown products of cholesterol. The liver also secretes cholesterol into the plasma in the form of lipoproteins. The major lipoprotein secreted by the liver in man appears to be very low-density lipoprotein (VLDL), which contains both triglyceride and cholesterol. The triglycerides of VLDL are removed in adipose tissue and the VLDL particle is ultimately converted into the cholesterol-rich lipoprotein, LDL. In normal humans, approximately 70”,, of the total cho-

Fig. 7. Model for plasma cholesterol transport in man. The physiologic implications of this model are discussed in the text. VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LCAT, lecithin: cholesterol acyltransferase.

1268

GOLDSTEIN

AND BROWN

lesterol in plasma is contained within the LDL particle. We envision that this circulating LDL acts as a storage bank for cholesterol. When certain extrahepatic cells, such as kidney cells, lung cells, blood cells, etc., require cholesterol for membrane synthesis, they behave in the same manner as do cultured fibroblasts and freshly isolated lymphocytes. That is, they synthesize LDL receptors, which allow them to bind and take up LDL and thus to use the lipoprotein to meet their own cholesterol requirements. As a result, in these extrahepatic parenchymal cells, the rate of cholesterol synthesis is kept low because the cells preferentially utilize the cholesterol contained in LDL. As the membranes of body cells turn over and as cells die and are renewed, cholesterol is released from tissues and enters the plasma. The important studies of Glomset suggest that the free cholesterol that leaves cells becomes associated in plasma with high-density lipoprotein (HDL), where it is converted to cholesteryl esters by the enzyme 1ecithin:cholesterol acyltransferase (LCAT).4’ The cholesteryl esters formed by the LCAT enzyme on the surface of HDL are eventually deposited in VLDL and then LDL. This establishes a cycle by which cells take up cholesterol from LDL and ultimately return the cholesterol to LDL. PATHOGENESIS FAMILIAL

OF THE

ATHEROSCLEROSIS

IN

HYPERCHOLESTEROLEMIA

Recent studies of the turnover of ‘251-LDL in human plasma by Bilheimer et al. in Dallas5’ and Simons et al. in Londor?’ can be interpreted to indicate that the specific LDL receptor-mediated process accounts for about two-thirds of the daily degradation of LDL in normal human subjects (see below). We believe that this constitutes the physiologic pathway for LDL degradation in the body. On the other hand, the LDL turnover studies also indicate that there is another mechanism for the degradation of LDL. Unlike the receptormediated degradation mechanism, the second mechanism functions in patients with homozygous familial hypercholesterolemia and so it does not depend on the LDL receptor. This finding has led us to propose that this second mechanism represents the uptake and degradation of LDL by scavenger cells that engage in nonspecific pinocytosis. In contrast to the receptor-mediated degradation pathway, the nonspecific scavenger cell pathway does not require the LDL receptor nor does it function to supply cholesterol to cells. Rather, it appears to be a pathway by which a constant 159, of plasma LDL is degraded daily in a manner analogous to the degradation of other plasma proteins such as albumin. It is likely that the bulk of LDL degradation by the scavenger pathway occurs in cells of the reticuloendothelial system. As an initial approach to explaining the consequences of an LDL receptor deficiency in familial hypercholesterolemia, we propose the hypothetical scheme in Fig. 8. This scheme, which is based on studies of the turnover of plasma ‘251-LDL in intact subjects, summarizes the quantitative aspects of the dual mechanism for degradation of plasma LDL as it exists in a 70-kg man. The assumptions that were made in developing this model are discussed in the legend to Fig. 8. As shown in Fig. 8A, in normal subjects who have a plasma LDL-cholesterol level of about 120 mg/dl, a total of 45% of the plasma LDLcholesterol pool is degraded daily. Of this, about 30”/, is cleared through the

1269

ATHEROSCLEROSIS

A NORMAL

PLASMA LDL-CHOLESTEROL

C

FH HOMOZYGOTE

-w

(mg/dll

mg/day

B

FH HETEROZYGOTE

I

-

CELL TYPE e

PARENCHYMAL

~~~~~

1 SCAVENGER CELLS

PARENCHYMAL _____

SCAVENGER CELLS

SCAVENGER CELLS

Fig. 8. Proposed model for the clearance of plasma LDL-cholesterol. The quantitative estimates are based on studies of the turnover of 12sl-LDL in the plasma of human subjects that have been performed by Longer et al.,52 Bilheimer et al.,5’J Simons et al.,51 and Packard et al.53 In general, these studies show that in normal subjects about 45% of the plasma LDL pool is cleared daily, whereas in familial hypercholesterolemia heterozygotes this value is about 25% and in homozygotes it is 15%. In using these data to develop the above model, we have made two major assumptions: (1) that the 15% clearance in the homozygote represents LDL cleared by receptorindependent pathways, and (2) that the same 15% clearance through the receptor-independent pathway occurs in all subjects and is independent of the plasma level of LDL. The latter assumption seems justified since the same 15% clearance of plasma LDL has been observed in a series of five familial hypercholesterolemia homorygotes whose plasma LDL-cholesterol levels varied from 390 to 910 mg/dl (Bilheimer, unpublished observations). Moreover, when the plasma LDLcholesterol level in one homozygote was reduced into the normal range by plasmaphoresis, the fractional clearance rate for LDL remained below 15% .63 The daily clearance of LDL-cholesterol (i.e., the sum of the absolute values for the clearance through the receptor-dependent and receptor-independent pathways) is highest in the homozygote (3000 mg), intermediate in the heterozygote (2000 mg), and lowest in the normal subjects ( 1500 mg). These numbers are within the range of the respective synthetic rates for LDL-protein as measured in steady-state turnover studies.50-53

LDL receptor mechanism and the released cholesterol is used physiologically by parenchymal cells for membrane synthesis, hormone synthesis, etc. This amounts to about 1000 mg of LDL-cholesterol per day. Although LDL receptors have not yet been shown on hepatocytes, it is likely that a major portion of the physiologic utilization may occur in hepatic cells, which may utilize LDL receptors to supply cholesterol for biliary excretion and for bile acid synthesis. In addition to these receptor-mediated pathways, 15”, of the plasma LDLcholesterol pool appears to be cleared by the receptor-independent mechanism. In normal subjects this scavenger mechanism clears about 500 mg of LDLcholesterol each day. Thus, the total LDL-cholesterol cleared each day in normal subjects averages about 1500 mg. 5o 53 The critical difference between the two mechanisms for clearing LDL lies in the fact that the receptor pathway is tightly controlled by the parenchymal cells themselves; that is, the receptor pathway will handle only enough cholesterol to meet the physiologic requirements of body cells. In effect, the feedback regulation of the LDL receptor places an upper limit on the amount of LDL that can be degraded through the physiologic pathway regardless of the level of plasma LDL. On the other hand, the scavenger pathway is not subject to feedback control, and as the plasma LDL level rises increasing amounts of LDL are degraded by this process.

1270

GOLDSTEIN

AND

BROWN

In patients with the heterozygous form of familial hypercholesterolemia, whose cells contain approximately 40% of the normal number of LDL receptors,54 the plasma LDL-cholesterol level rises to 24 times above normal, to 300 mg/dl (Fig. 8B). Under these conditions, the high concentration of plasma LDL compensates for the reduced number of receptors, so that a nearly normal amount of LDL can be degraded through the receptor pathway daily.54.55 Thus, a near-normal amount of cholesterol can be delivered in a physiologic manner to parenchymal cells. However, because the receptor deficiency elevates the concentration of plasma LDL in heterozygotes and because the clearance by scavenger cells represents 15”,/ of the LDL pool no matter what the concentration of plasma LDL, the absolute amount of LDL degraded by the scavenger pathway in heterozygotes is about 2$ times above normal. In patients with homozygous familial hypercholesterolemia, this situation is carried to its extreme. As shown in Fig. 8C, the physiologic LDL receptormediated pathway is abolished. As a result, the plasma LDL-cholesterol level rises to extremely high values. The 157, of the plasma LDL degraded through the scavenger mechanism each day amounts to approximately 3000 mg of LDLcholesterol.50*5’ This represents an enormous load of cholesterol that must be cleared each day by the scavenger cells. When the LDL level is elevated in plasma, whether due to a genetic deficiency in LDL receptors as in familial hypercholesterolemia or due to other genetic and/or environmental factors, cholesteryl esters deposit in the artery wall and atherosclerosis ultimately develops. We suggest that the smooth muscle cells in the atherosclerotic lesion behave functionally like the scavenger cells shown in Fig. 8; that is, when endothelial damage occurs and plasma leaks into the artery wall, the smooth muscle cells function to take up and digest LDL by a process that does not involve the LDL receptor. In this sense, the behavior of smooth muscle cells in vivo resembles the behavior of cells in culture when presented with polycationic DMPApLDL.j9 In both cases, the cells accumulate massive amounts of cholesteryl esters until cellular toxicity results. LDL RECEPTOR

HYPOTHESIS

The most logical conclusion from the above studies is that the receptormediated LDL pathway functions in man to protect against atherosclerosis. This protection is achieved through two properties of the cell-surface LDL receptor. First, as discussed above, the LDL receptor is subject to feedback regulation. This permits most cells to suppress LDL receptor activity when cholesterol stores are adequate, thus avoiding the type of pathologic overaccumulation of cholesteryl esters that occurs in atherosclerosis. Second, the LDL receptor binds LDL with high affinity. This high affinity allows the cells to satisfy all of their cholesterol requirements under conditions in which the level of LDL in plasma and in interstitial fluid is kept low. Thus, the LDL receptor allows the body to solve its dual problemthat is, it can maintain plasma LDL levels below the threshold range for athersclerosis and at the same time supply its cells with adequate amounts of cholesterol. That the LDL receptor functions both to keep the plasma LDL level low and to protect against atherosclerosis is nowhere more evident than in the data illustrated in Fig. 9. Normal subjects, whose fibroblasts can develop about

1271

ATHEROSCLEROSIS

Fig. 9. typical function blasts with

Concentration

age

of the derived

number from

lated

from

subjects

and

per in

measured

that

on

and

the as a

flbro-

subjects forms

of

hypercholesterolemia.

experiments was

plotted

homozygous

familial

fibroblasts

LDL and

of LDL receptors

of LDL receptors

lz5 I-LDL binding growing

plasma

infarction

normal

the heterozygous

receptor-negative The number

of

at myocardial

were

cell

was

which

calcu-

maximal

at 4°C

in actively

deprived

of LDL for

ILDLRECEPTORS ON (number

48 hr.14

FIBROBLASTS x 10 3/ce11l

10,000 LDL receptors per cell, maintain a plasma LDL-cholesterol level of about 120 mg/dl and are generally protected against myocardial infarction for about 70 yr. Subjects with the heterozygous form of familial hypercholesterolemia, whose fibroblasts can develop only about 40”:, of the normal number of LDL receptors, must maintain a plasma LDL-cholesterol level that is 2i times above normal and in general develop myocardial infarctions 20 yr earlier than do normal subjects. Subjects with the homozygous form of familial hypercholesterolemia, whose fibroblasts lack LDL receptors, maintain plasma LDLcholesterol levels that are at least sixfold higher than normal, and as a result they usually develop myocardial infarctions before age 20.24m’6 THE

“NORMAL” MAY

LEVEL BE

OF

PLASMA

UNPHYSIOLOGICALLY

LDL

IN

WESTERN

MAN

HIGH

If the genetic defect in LDL receptors can explain the premature occurrence of atherosclerosis in familial hypercholesterolemia patients, what mechanism can be invoked to explain the nearly universal occurrence of atherosclerosis at later ages in “normal” Western man? As discussed above, the body seeks to minimize the leakage of LDL into the artery wall by adjusting the plasma LDL concentration to the lowest level at which the lipoprotein can function to supply cholesterol to cells. The high-affinity LDL receptor permits such an adjustment to occur. However, despite the fact that normal Western man possesses such a receptor, his plasma LDL level is considerably higher than the level required to achieve adequate binding to his own LDL receptor, at least as measured in vitro. Thus, in all human cells so far studied (i.e., fibroblasts, arterial smooth muscle cells, cultured lymphoid cells, and circulating lymphocytes), the LDL receptor is capable of supplying sufficient cholesterol to support ceil growth and keep HMG CoA reductase suppressed when the lipoprotein is present at a concentration of about 25 pug of LDL-cholesterol/ml.5~7,4’ 47 Other than blood cells and endothelial cells, most body cells cannot obtain LDL from plasma directly, but rather must obtain it from the ultrafiltrate of plasma that constitutes the intersitital fluid. Because of the large size of the LDL particle, the capillary endothelium serves as a major barrier to its passage into the interstitial fluid and hence the concentration of LDL in interstitial fluid is much lower than that in plasma. Although a precise quantitation of the

1272

GOLDSTEIN

AND

BROWN

level of LDL in the interstitial fluid of man is not yet available, estimates of the total cholesterol content of human peripheral lymph fluid have been made by Reich1 and co-workers, who found that the concentration of sterol in lymph was about one-tenth that in plasma. 56 Moreover, the cholesterol content in lymph (which approximates the interstitial fluid) was significantly correlated with the plasma LDL-cholesterol concentration.56 These workers also found that 14 hr after the intravenous injection of ‘)‘I-LDL into two human subjects the concentration of protein radioactivity in the lymph was about 10% of that in plasma.57 Considered together, the above data suggest that the concentration of LDL in the interstitial fluid of man is of the order of 10% of that in plasma. In view of the data suggesting that the LDL receptor functions optimally at LDL levels of about 25 pg LDL-cholesterol/ml (i.e., 2.5 mg/dl) and in view of the ten-fold gradient in LDL concentration between plasma and interstitial fluid, the appropriate plasma LDL-cholesterol level in man should be about 25 mg/dl. However, in normal Western man, the mean level of plasma LDL-cholesterol is about 120 mg/d1.26 Assuming that the LDL receptor functions in the body as it does in cultured cells, one must conclude that the mean plasma LDL-cholesterol level in normal man is about five-fold higher than the level required for plasma LDL to deliver cholesterol to body cells. It is significant to note that the postulated “appropriate” level of LDLcholesterol in man (25 mg/dl) is similar to the mean level of LDL-cholesterol (32 mg/dl) measured in eight other mammalian species that are not naturally susceptible to atherosclerosis.58 Moreover, this “appropriate level” is only slightly below the mean level ofLDL-cholesterol (30 mg/dl) observed in normal human newborn infants at a time before dietary and other environmental stresses have elevated it.59 Thus, the data on the LDL pathway lend biochemical support to the conclusion already derived from epidemiologic studies: namely, that the level of plasma LDL-cholesterol in “normal” Western man is too high.60-62 From the above discussion, it seems clear that the levels of LDL in plasma and interstitial fluid observed in Western man after the first year of life are several-fold above the levels necessary for LDL to bind to the LDL receptor and deliver cholesterol to body cells. We suggest that these populations are concentration of plasma LDL prone to atherosclerosis because their “normal” is so unphysiologically high that when plasma leaks into the artery wall through areas of endothelial damage, the smooth muscle cells are presented with a load of LDL that exceeds the clearance capacity of the receptor-mediated process. As a result of this overload, LDL is taken up by a receptor-independent scavenger process that leads to an uncontrolled accumulation of cholesteryl esters within smooth muscle cells. By this formulation, it is the presence in Western man of a plasma LDL level above that required to saturate the LDL receptor that renders this segment of the human species uniquely susceptible to atherosclerosis. REFERENCES 1. Smith EB, Smith aortic

itima,

in Paoletti

RH: Early changes in R, Gotto AM Jr (eds):

Atherosclerosis Reviews, Raven, 1976, p I19

vol.

1. New

York,

1273

ATHEROSCLEROSIS

2. Wissler RW: Development of the atherosclerotic plaque, in Braunwald E (ed): The Myocardium: Failure and Infarction. New York. H.P. Publishing, 1973, p 155 3. Ross R, Glomset JA: The pathogenesis of atherosclerosis. N Engl J Med 295:369, 420. 1976 4. French JE: Atherosclerosis in relation to the structure and function of the arterial intima, with special reference to the endothelium. Int Rev Exp Pathol 5:253, 1966 5. Goldstein JL, Brown MS: The low density lipoprotein pathway and its relation to atherosclerosis. Ann Rev Biochem 46:897. 1977 6. Brown MS, Goldstein JL: Receptormediated control of cholesterol metabolism. Science 191:150, 1976 7. Goldstein JL, Brown MS: The LDL pathway in human fibroblasts: A receptormediated mechanism for the regulation of cholesterol metabolism, in Horecker BL. Stadtman ER (eds): Current Topics in Cellular Regulation, vol 2. New York, Academic, 1976, p 147 8. Brown MS. Goldstein JL: Familial hypercholesterolemia: A genetic defect in the lowdensity lipoprotein receptor. N Engl J Med 294: 1386. 1976 9. Jackson RL. Morrisett JD, Gotto AM Jr: Lipoprotein structure and metabolism. Physiol Rev 56:259. 1976 IO. Goodman DS: Cholesterol ester metabolism. Physiol Rev 45:747, 1965 11. Anderson RGW, Goldstein JL, Brown MS: Localization of low density lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc Natl Acad Sci USA 73:2434, 1976 12. Brown MS, Goldstein JL: Familial hypercholesterolemia: Defective binding of lipoproteins to cultured fibroblasts associated with impaired regulation of 3-hydroxy-3methylglutaryl coenzyme A reductase activity. Proc Nat1 Acad Sci USA 71:788, 1974 13. Goldstein JL. Brown MS: Binding and degradation of low density lipoproteins by cultured human fibroblasts: Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem 249:5153. 1974 14. Goldstein JL, Basu SK, Brunschede GY, et al: Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans. Cell 7:85, 1976 15. Brown MS, Goldstein JL: The low density lipoprotein pathway in human fibroblasts. Trends Biochem Sci 1:193, 1976

16. Goldstein JL, Brunschede GY, Brown MS: Inhibition of the proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. J Biol Chem 250:7854, 1975 17. Brown MS, Dana SE. Goldstein JL: Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein. Proc Nat] Acad Sci USA 72:2925, 1975 18. Goldstein JL, Dana SE. Faust JR, et al: Role of lysosomal acid lipase in the metabolsim of plasma low density lipoprotein: Observations in cultured fibroblasts from a patient with cholesterol ester storage disease. J Biol Chem 250:8487, 1975 19. Brown MS, Faust JR, Goldstein JL: Role of the low density lipoprotein receptor in regulating the content of free and esterified cholesterol in human fibroblasts. J Clin Invest 55:783, 1975 20. Brown MS, Goldstein JL: Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-keto-cholesterol. J Biol Chem 249:7306, 1974 21. Brown MS, Dana SE, Goldstein JL: Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human fibroblasts: Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem 249:789, 1974 22. Goldstein JL, Dana SE, Brown MS: Esterification of low density lipoprotein cholesterol in human fibroblasts and its absence in homorygous familial hypercholesteroiemia. Proc Natl Acad Sci USA 71:4288, 1974 23. Brown MS, Dana SE. Goldstein JL: Cholesterol ester formation in cultured human fibroblasts: Stimulation by oxygenated sterols. J Biol Chem 250:4025, 1975 24. Khachadurian AK: The inheritance essential familial hypercholesterolemia. J Med 37:402, 1964

of Am

25. Brown MS, Goldstein JL: Familial hypercholesterolemia: Genetic, biochemical and pathophysiologic considerations. Adv Intern Med 20:273. 1975 26. Fredrickson DS, Goldstein JL. Brown MS: The familial hyperlipoproteinemias, in Stanbury JB. Wyngaarden JB. Fredrickson DS (eds): The Metabolic Basis of Inherited Disease (ed 4). New York, McGraw-Hill, 1978. p 604 27. Goldstein JL, Schrott HG, Hazzard WR, et al: Hyperlipidemia in coronary heart disease: II. Genetic analysis of lipid levels in 176

1274

families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest 52:1544, 1973 28. Stein 0, Weinstein DB, Stein Y, et al: Binding, internalization, and degradation of low density lipoprotein by normal human fibroblasts and by fibroblasts from a case of homozygous familial hypercholesterolemia. Proc Natl Acad Sci USA 73:14, 1976 29. Goldstein JL. Brown MS: Familial hypercholesterolemia: Identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol. Proc Natl Acad Sci USA 70:2804. 1973 30. Khachadurian AK, Kawahara FS: Cholesterol synthesis by cultured fibroblasts: Decreased feedback inhibition in familial hypercholesterolemia. J Lab Clin Med 83:7, 1974 3 1. Goldstein JL, Dana SE, Brunschede GY. et al: Genetic heterogeneity in familial hypercholesterolemia: Evidence for two different mutations affecting functions of low-density lipoprotein receptor. Proc Natl Acad Sci USA 72: 1092, 1975 32. Brown MS, Goldstein JL: Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptor-bound low density lipoprotein. Cell 9:663, 1976 33. Goldstein JL, Brown MS, Anderson RGW: The LDL pathway in human fibroblasts: Biochemical and ultrastructural correlations, in Brinkley BR, Porter KR (eds): International Cell Biology 1976-1977. New York. Rockefeller University Press, 1977 (in press) 34. Roth TF, Porter KR: Yolk Protein uptake in the oocyte of the mosquite Aedes Aegypti. I. J Cell Biol 20:313, I964 35. Fawcett DW: Surface specializations of absorbing cells. J Histochem Cytochem 13:75, 1965 36. Korn ED: Biochemistry of endocytosis, in Fox CF (ed): MTP International Review of Science, Vol. 2. London, Butterworths, 1975. 37. Anderson RGW, Brown MS, Goldstein JL: Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human Iibroblasts. Cell lo:35 1, 1977 38. Brown MS, Goldstein JL: Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Cell 6:307, 1975 39. Goldstein JL, Anderson RGW, Buja ML, et al: Overloading human aortic smooth muscle cells with low density lipoprotein cholesteryl esters reproduces features of atherosclerosis in vitro. J Clin Invest. June, 1977 (in press)

GOLDSTEIN

AND

BROWN

40. Basu SK, Goldstein JL, Anderson RGW, et al: Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Nat1 Acad Sci USA 73:3178, 1976 41. Basu SK, Anderson RGW. Goldstein JL, et al: Metabolism of cationized lipoproteins by human fibroblasts: Biochemical and morphologic correlations, J Cell Biol, July, 1977 (in press) 42. Goldstein JL, Brown MS: Lipoprotein receptors, cholesterol metabolism. and atherosclerosis. Arch Pathol 99: I8 I, 1975 43. Bierman EL, Albers JJ: Lipoprotein uptake by cultured human arterial smooth muscle cells. Biochim Biophys Acta 388:198. 1975 44. Kayden HJ, Hatam L. Beratis NC: Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and the esterification of cholesterol in human long term lymphoid cell lines. Biochemistry l5:521, 1976 45. Ho YK, Brown MS, Kayden HJ, et al: Binding, internalization, and hydrolysis of low density lipoprotein in long-term lymphoid cell lines from a normal subject and a patient with homozygous familial hypercholesterolemia. J Exp Med 144:444, 1976 46. Ho YK, Brown MS, Bilheimer DW, et al: Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes. J Clin Invest 58: 1465, 1976 47. Ho YK, Faust JR, Bilheimer DW. et al: Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes: Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia. J Exp Med. June, 1977 (in press) 48. Have1 RJ: Lipoproteins and lipid transport, in Kritchevsky D, Paoletti R, Holmes WL (eds): Advances in Experimental Medicine and Biology. Vol. 63. New York, Plenum, 1975. P 37 49. Glomset JA: The cholesterol acyltransferase Res9:155, 1968

plasma reaction.

lecithin: J Lipid

50. Bilheimer DW. Goldstein JL. Grundy SM. et al: Reduction in cholesterol and low density lipoprotein synthesis after portacaval shunt surgery in a patient with homozygous familial hypercholesterolemia. J Clin Invest 56: 1420, 1975 51. Simons LA, Reich1 D, Myant The metabolism of the apoprotein low density lipoprotein in familial

NB, et al: of plasma hyperbeta-

1275

ATHEROSCLEROSIS

lipoproteinemia in the homozygous form. Atherosclerosis 21:283, 1975 52. Langer T, Strober W. Levy RI: The metabolism of low density lipoprotein in familial type II hyperlipoproteinemia. J Clin Invest 51:1528, 1972 53. Packard CJ, Third JLHC, Shepard J, et al: Low density lipoprotein metabolism in a family of familial hypercholesterolemic patients. Metabolism 25:995, 1976 54. Brown MS. Goldstein JL: Expression of the familial hypercholesterolemia gene in heterozygotes: Mechanism for a dominant disorder in man. Science 185:61, 1974 Brown MS: Familial 55. Goldstein JL, hypercholesterolemia: A genetic regulatory defect in cholesterol metabolism. Am J Med 58: 147. 1975 56. Reich1 D. Simons LA, Myant NB, et al: The lipids and lipoproteins of human peripheral lymph, with observations on the transport of cholesterol from plasma and tissues into lymph. Clin Sci Mol Med 45:313, 1973

57. Reich1 D, Postiglione A, Myant NB. et al: Observations on the passage of apoproteins from plasma lipoproteins into peripheral lymph in two men. Clin Sci Mol Med 49:419, 1975 58. Mills GL, Taylaur CE: The distribution and composition of serum lipoproteins in eighteen animals. Comp Biochem Physiol 408: 489,197l 59. Tsang RC, Fallat RW, Glueck CH: Cholesterol at birth and age I: Comparison of normal and hypercholesterolemic neonates. Pediatrics 53:458, 1974 60. Keys A: Coronary heart disease: The global picture. Atherosclerosis 22: 149, 1975 61. Stamler J: Epidemiology of coronary heart disease. Med Clin North Am 57:s. 1973 62. Keys A: Coronary heart countries. Circulation 4l(Suppl

disease in seven I): 1, 1970

63. Thompson CR, Myant N: Low density lipoprotein turnover in familial hypercholesterolemia after plasma exchange. Atherosclerosis 23:371. 1976