Platelets: Cell Proliferation and Atherosclerosis

Russell Ross

lntimal smooth muscle proliferation is the hallmark of the lesions of atherosclerosis. Endothelial injury is postulated to precede this intimal smooth muscle proliferative response, which is mediated by a potent mitogenic factor derived from adherence, aggregation, and release by platelets at sites of endothelial injury. Smooth muscle proliferation is accompanied by varying amounts of connective tissue formation and intracellular and extracellular lipid deposition, dependent upon the risk factors encountered in each patient. The platelet-derived mitogen (PF) is a stable, cationic, relatively low molecular weight ~10.000-30.000) protein that has been partially purified by ion exchange chromatogra- phy and gel filtration. Less than 100 ng of PF/ml culture medium can stimulate sparse 3T3 cells or smooth muscle cells, but not endothelial cells, to undergo multiple cell divisions in the presence of

5% cell-free, plasma-derived serum. The latter

contains no mitogenic activity. The interaction of

the platelet mitogen and plasma-derived compo-

nents, including lipoproteins, plays a critical role in

smooth muscle proliferation in vitro and in vivo in

the induction of the lesions of atherosclerosis.

I T IS NOW A GENERALLY accepted maxim that the principal development that

leads to the lesions of atherosclerosis is an inti- ma1 proliferation of smooth muscle cells. As each lesion develops, it becomes increasing complex and takes on different aspects of three phenom- ena. These are: (1) intimal smooth muscle prolif- eration, (2) formation of new connective tissue (including collagen, glycosaminoglycan, and elastic fibers), and (3) increased deposition of both intracellular and extracellular lipids.’

The different lesions of atherosclerosis that have been described in man include the fatty streak, the fibrous plaque, and the so-called

From the Department of Pathology, School of Medicine, University of Washington, Seattle. Wash.

Presented at the Pfizer Biomedical Research Sympo- sium-progress Toward Understanding and Treating Diabetic Complications, Branford House, Groton. Corm.. September 18-19, 1978.

Supported in part by USPHS Grants AM 13970. HL18645. and AG00299, Grant RP-00266 from the Regional Primate Center, and a grant from R. J. Reynolds Industries, Inc.

Address reprint requests to Russell Ross, M.D., Depart- ment of Pathology, School of Medicine, University of Wash- ington, Seattle. Wash. 98195.

a1979 by Grune & Stratton. Inc. 00260495/79/2813-0004$01.00/0

410

complicated lesion. Each of these contains different mixtures of the three phenomena. For example, in the fatty streak there are relatively small numbers of proliferated cells so that the lesion does not greatly thicken the intima, but rather causes a yellow discoloration due to the extensive deposits of lipid that occur throughout the lesion. When examined by light and electron microscopy the fatty streak consists principally of intracellular accumulations of lipid in smooth muscle cells and in macrophages. In contrast, the fibrous plaque can produce a marked increase in intimal thickness, and contains a large number of proliferated smooth muscle cells that have formed new connective tissue. The deeper part of the fibrous plaque often contains cell debris and lipids and is covered by a cap of relatively dense connective tissue. The complicated lesions repre- sent fibrous plaques, which have taken on an altered appearance and often contain large calcified deposits. The fibrous plaque and the complicated lesions are generally responsible for the principal clinical sequelae of atherosclerosis, myocardial and cerebral infarction.

The realization that intimal smooth muscle cell proliferation represents a key event in under- standing the genesis of the lesions of atheroscle- rosis has led to increasing interest and research on the part of a number of investigators in examining those factors that are associated with the smooth muscle proliferative response. If it were possible to prevent this proliferative response, it should be possible to prevent the principal lesions and, thus, the important clinical sequelae. This realization has provided new opportunities to study the smooth muscle pro- liferative response at both the cellular and the molecular level. Many interesting correlative in vitro and in vivo studies have resulted from this approach.

PLATELETS AND SMOOTH MUSCLE CELL PROLIFERATION IN CULTURE

Techniques to grow arterial cells in culture have developed rapidly in the last decade. At the same time, it has been possible to show that the proliferative response that results from exposing cells to serum in culture is due principally to a

Metabolism, Vol. 28. No. 4, Suppl. 1 (April), 1979

PLATELETS AND ATHEROSCLEROSIS 411

IO' 7 M57 ThA

T, 4-7-75

5% PDS t2x Plot&t Release

.I 5% PDS t Platelet Release FracIU

,,,I-----+---___ -I 5% PDS

-----I l/Z% WBS

I IO4 i

0 I 2 3 4 5 6 7 8 9 IO II 12 I3 14

Days In culture

Fig. 1. Growth of monkey aortic smooth muscle C8& in plasma-derived Serum plus platelet factor. Cells Were plated on day 0 in 35-mm dishes in 0.5 % monkey whole blood serum. On day 4, the medium was changed and the various test media were added. Two plates per group were trypsinized and counted each day. Medium was changed every 2 days. The 2X platelet release is material obtained from thrombin-treated human platelets. Platelet release fraction 111 is platelet raleasate that has been partially purified by carboxymethyl Sephadex chromatography. (WBS) Whole blood serum: (PDSJ plasma- derived serum. (Reproduced with permission from celfl”

mitogenic factor present in whole blood serum

that is derived from platelets.‘-’

The experiments that demonstrated that the

platelets provide a growth factor that represents the principal mitogen present in whole blood serum were performed using monkey whole

blood serum, cell-free, plasma-derived serum, and cell-free, plasma-derived serum containing

material released from platelets. Each of these was studied in terms of their mitogenic effect upon arterial smooth muscle cells, arterial endo- thelial cells, fibroblasts, and several other cell types in terms of their proliferative response in

culture.

Figure 1 demonstrates some of the observa-

tions from this study. In this figure, it can be

seen that smooth muscle cells are quiescent in cell-free, plasma-derived serum (PDS), as they

are quiescent in very low concentrations of whole

blood serum. When a crude preparation of mate-

rial released from a purified preparation of

platelets is added to the PDS, the proliferative capacity of this plasma-derived serum is restored to an extent equivalent to that of whole blood serum. Further, a semi-purified preparation of the material released from the platelets (obtained by chromatography on CM-Sephadex and entitled CMS III) also restores this activity.

412 RUSSELL ROSS

Table 1. Characteristics of the Platelet-Derived

Growth Factor

Basic

pl9.5-10.4

Molecular weight

10.000-30.000 by gel filtration

25.000-32.000 by elution from SDS gels

Stable under conditions

6 M Gu HCI 2% SDS

B M Urea PH 4

Heat 56°C for 30 min

Activiry destroyed by

Trypsin

by reduction and alkylation with DTT/

lodoacetamide

Hydophobic-strongly adsorbed to phenyl sepharose

No somatomedin-C activity by RIA

The results of these studies taken together with a further series of studies using more purified preparations of the platelet-derived growth factor (PF) have demonstrated that this factor is the principal mitogen in whole blood serum that stimulates those cells that require serum to pro- liferate in culture.

Several laboratories have been examining the nature of this platelet-derived growth factor in a series of experiments in which the factor has been isolated, further purified, and is in the process of being characterized. The studies to date demonstrate the following characteristics for the platelet-derived growth factor. (Table 1).

Thus, the platelet-derived growth factor is a highly stable, cationic protein of relatively low molecular weight that is very active at low concentrations (< 100 ng/ml culture medium of semi-pure material) when exposed to most cells in culture.

PLATELETS AND ENDOTHELIAL CELLS

IN CULTURE

An important exception is the observation that arterial endothelial cells do not require the platelet-derived growth factor to proliferate in culture. The endothelium is able to proliferate in the presence of cell-free, plasma-derived serum as effectively as in whole blood serum or plasma- derived serum containing the platelet factor. This observation will undoubtedly provide important clues concerning further understand- ing of the factors required for the maintenance of endothelial integrity, both in vivo and in cell culture, since maintenance of endothelial integ- rity is critical in the prevention of atherogenesis.

PLATELETS AND PLASMA PLAY

A COOPERATIVE ROLE

Having observed that the platelet-derived growth factor is a potent mitogen, Vogel et a1.6 examined the cooperative role that plasma plays together with the platelet factor in permitting cells to undergo DNA synthesis and divide in culture. These studies demonstrated that a mini- mal amount of plasma is necessary in the culture medium for cells to traverse the cell cycle and divide. The principal role played by the platelet- derived growth factor is to induce DNA synthe- sis.‘-” Plasma molecules are absolute require- ments for cells to further traverse the cell cycle, undergo cell doublings, and increase in number.6 Cells in vivo are bathed in either plasma (endo- thetium) or filtrates of pIasma (all other cells). They are only exposed to the equivalent of whole blood serum when there is injury and blood coagulation. Whole blood serum, by definition, includes material released from blood platelets during the process of coagulation together with constituents present in plasma. This may, from a teleologic point of view, explain why quiescent cells in vivo may be similar to cells given 5% or 10% cell-free, plasma-derived serum in culture, since plasma-derived serum would lack any platelet mitogens.” Conversely, cells given whole blood serum in culture would respond as they do in vivo when platelet products are released to help in the initiation of cell proliferation.

CORRELATIVE STUDIES OF ATHEROSCLEROSIS

IN VW0

It is important to determine whether or not mitogens derived from platelets actually play a role in the development of the lesions of athero- sclerosis in vivo.

Several studies have demonstrated that plate- lets do, in fact, play a key role in atherogenesis. This was shown by demonstrating that platelet function must be intact to be able to induce the intimal proliferative lesions of experimental atherosclerosis.

Harker, et al.12 were able to produce extensive lesions of atherosclerosis by inducing chronic homocysteinemia in the baboon. In this experi- mental model, chronic homocysteinemia leads to focal sites of altered arterial endothelial cells, These sites contain missing and altered cells and are accompanied by a marked decrease in the

PLATELETS AND ATHEROSCLEROSIS 413

survival of “Cr-labeled autologous platelets in each of the homocysteinemic animals. The decrease in platelet survival suggests that the platelets are continuously activated in relation to the injured endothelium and then removed from the circulation. After 90 days of chronic homo- cysteinemia and continued decreased platelet survival, the endothelial injury is accompanied by the development of marked intimal smooth muscle proliferative lesions of atherosclerosis in the baboons equivalent to fibrous plaques in

man. When Harker and his colleagues interfered with platelet function with the pharmacologic agent, dipyridamole (an agent that prevents platelet adherence and release), platelet survival returned to normal levels and they were able to completely inhibit the intimal smooth muscle proliferative response and thus prevented the formation of lesions.

Similarly, Moore et al.” used an anti-platelet serum to induce a thrombocytopenia in rabbits. In the absence of circulating platelets, they

Fig. 2. In the response to injury hypothesis, two different cyclic events may occur. The outer, or regression cycle. may represent common single occurrences in all individuals in which endothelial injury leads to desquamation, platelet adherence, aggregation, and release, followed by intimal smooth muscle proliferation and connective tissue formation. If the injury is a single event. the lesions may go on to heal and regression occur. The inner or progression cycle demonstrates the possible consequences of repeated or chronic endothelial injury as may occur in chronic hyperlipidemia. In this instance. lipid deposition as well as continued smooth muscle proliferation may occur after recurrent sequences of proliferation and regression, and these may lead to complicated lesions that calcify. Such lesions could go on to produce clinical sequelae such as thrombosis and infarction. (Reproduced with permission from Science).”

414 RUSSELL ROSS

prevented the development of experimentally induced lesions with either a chronic indwelling catheter or after deendothelialization with an intraarterial balloon catheter.14

Fuster et al.” demonstrated that it was possi- ble to prevent the development of experimental atherosclerosis in swine homozygous for von Willebrand’s disease. In this particular genetic disease, factor VIII is missing from the plasma and platelets are unable to adhere, and undergo, the release reaction. In matched normal and von Willebrand swine, both of which were hypercho- lesterolemic as a result of high cholesterol diets, Fuster and his colleagues produced extensive lesions of atherosclerosis in the normal hyper- cholesterolemic swine, whereas the hypercholes- terolemic swine with von Willebrand’s disease had essentially no lesions.15

in the endothelium may be subtle and be mani- fest only as changes in permeability or they may be obvious resulting in focal desquamation and in loss of cells. At such sites of altered endothe- lium platelets may adhere, aggregate, and release their constituents. At the same time, plasma components may enter the artery wall and act with platelet products on the intimal smooth muscle cells inducing the smooth muscle migration and proliferation found in the result- ing lesion. The hypothesis suggests that if the lesions are single events, the proliferative response may be reversible and no clinical seque- lae will occur. On the other hand, if the injury continues on a chronic or recurrent basis, the lesions may progressively increase in size until clinical sequelae occur (Fig. 2) .I6

THE RESPONSE TO INJURY HYPOTHESIS

These studies provide additional data in rela- tion to the “Response to Injury Hypothesis” being tested in our laboratory and in several other laboratories. This hypothesis suggests that the lesions of atherosclerosis begin as a result of some form of endothelial alteration. Alterations

The lesions of atherosclerosis are complex lesions related to a number of different risk factors. This hypothesis will provide an opportu- nity to examine the potential role of each of these factors in relation to the etiology and pathogene- sis of atherosclerosis, and in relation to improv- ing our understanding of the role of the platelet- derived growth factor and constituents in plasma in the genesis of this important disease process.

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