Cellular processes in atherogenesis: Potential targets of Ca2+ channel blockers

Arhrrosclerosk, 88 (1991) 109-132 0 1991 Elsevier Scientific Publishers ADONIS 0021915091001163

ATHERO

109 Ireland,

Ltd. 0021.Yl5O/Y1/$03.50

04652

Review article

Cellular processes in atherogenesis: potential targets of Ca2’ channel blockers G. Schmitz, J. Hankowitz, and E.M. Kovacs Institut fib Klinische Chemie und Lahorutoriumsmedizin

Westfii’lischr Wilhelms-Unlr,ersitiit,

Miinster (Germany)

(Received 14 January, 1091) (Revised, received 8 March. 19Y1) (Accepted 13 March. 1991)

Summary

Atherosclerosis is characterized by increased endothelial permeability, monocyte infiltration, intimal smooth muscle cell @MC) proliferation, platelet aggregation and the accumulation of lipids, calcium and extracellular matrix components in the vessel wall. In various animal studies and recently in humans it could be established that Ca2+ channel blockers delayed the progression of the atherosclerotic process at the stage of early lesions. This review surveys the interaction of Ca2+ channel blockers with various membrane proteins (purinergic receptors, nucleoside transporter, peripheral benzodiazepine receptors, multi-drug resistance protein) which are involved in signal transduction and their potential impact on the observed antiatherosclerotic effects. Although the precise mechanisms have yet to be fully elucidated, it has been clearly shown that these drugs inhibit smooth muscle cell proliferation and migration, improve cellular lipoprotein metabolism in vascular cells, alter phospholipid turnover, decrease platelet adhesion in the vessel wall, reduce extracellular matrix synthesis and protect against radical induced cell damage. Most of these effects are independent of Ca*+ flux across voltage-operated Ca*+ channels. However, all these processes are relevant to the pathogenesis of atherosclerosis and therefore the elucidation of the antiatherogenic mechanisms of Ca2+ channel blockers at the cellular level is of great interest. The future development of Ca2+ channel blockers with altered molecular structures optimized for their antiatherosclerotic targets may provide a useful tool in the therapy of atherosclerosis and risk factor intervention. The protective mechanisms are related to a stabilization of cell membrane integrity, the

Correspondence to: Prof. G. Schmitz, Institut fiir Klinische Chemie und Laboratoriumsmedizin, Westfilische WilhelmsUniversitit, Albert-Schweitzer-Stral3e 33, W-4400 Miinster. Germany. Fax: 0251/83 6960

110

modulation of secretory activities and cell/cell plasma lipoprotein levels.

Key words: Atherosclerosis; metabolism

Ca2+ channel

communication

blocker;

Signal transduction;

Membrane

integrity;

Lipid

eration, disturbances in platelet function and fibrinolysis (Fig. 1). With our current knowledge the modified “response to injury” hypothesis provides a plausible explanation [ 1,2] where atherosclerosis appears to be induced by alterations of the endothelial cells. At the early stage of atherosclerosis the initial dysfunction of endothelial cells in response to an

Current concepts of atherosclerotic mechanisms at the cellular level and potential targets for Ca*+ channel blockers Atherosclerosis can be defined as an intimal disorder of the vessel wall which includes endothelial damage, subendothelial lipid and Ca2+ accumulation, intimal smooth muscle cell prolif-

-Lng

processes rather than to a lowering of

Stimuli ( $iEia

*

e Inflammatory

1

response

Increased endothelial replication and permeability

Infiltration ot macrophages

modified

IlpOprOteinS b

phagocytoslng

Ma

\

‘I

PDGF

collagen elastin proteoglycans

??

secret’o”

1

activated I 4

Elastica

Interna

M 0

/I

PDGF

migration

c(

-

llke protein 12. HETE prostanotds

Fig. 1. Modified “response to injury” hypothesis. The intact endothelium in response to an irritating stimuli induces increased endothelial replication and permeability, intimal oedema and infiltration of macrophages. EC modified lipoproteins are released to the subendothelial space and phagocytosed by macrophages which transform into foam cells. Due to the multilateral cellular interactions activated cells produce mitogens for vascular SMC which migrate from the media into the intima, proliferate and transform into a synthetic phenotype with the characteristics of enhanced extracellular matrix formation.

111 irritating stimulus such as hypercholesterolemia, immune activation, hypertension, anoxia or turbulent blood flow without morphological signs of endothelial denudation seems to be the major event [3]. The response of the irritated endothelial cells induces an increase in permeability for plasma components into the subendothelial space associated with the enhanced infiltration of monocytes and enhanced endothelial cell replication and turnover. In the subendothelium monocytes differentiate into macrophages which ingest large amounts of cholesterol leading to the formation of foam cells [4,5]. At this stage macrophages secrete a number of products influencing further chemotaxis, vascular permeability and endothelial cell integrity. Therefore the morphologically visible injury of the endothelial cells is rather considered to be a later event with the consecutive modulation of thrombogenesis and

0A

fibrinolysis. Due to several cellular interactions within the injured vessel wall endothelial cells, macrophages and platelets release growth factors which are chemotactic and mitogenic for vascular SMC. Vascular SMC are assumed to migrate from the media to the intima where they convert from a contractile into a synthetic phenotype, proliferate and secrete growth factors and extracellular matrix components [6]. These events are mediated by various cellular interactions via cell surface receptors causing changes in cellular signal transduction pathways. Impaired functions atherosclerosis

of

endothelial

cells

in

The early response of endothelial cells to an irritating stimulus is the regulation of the vascular tone. PGI, is an important vasodilatator pro-

0B

Platelet/endothelial

interaction during coagulation

Regulation of fibrinolris on the surface of endothe ium

Plasmlnogen TXA,

b

Plasmin

___) I FDP

PAIL1 1 1

T 1~PA -

I # ‘t

x

-

Endothelial cell

Endothelln (EDCF)

I

SMC SMC Fig. 2. A: Interaction between platelets and endothelial ceils during coagulation. Platelets adhere to the injured endothelium. Subendothelium has a highly reactive surface to promote platelet adhesion and aggregation with its collagen, proteoglycan, glycoprotein and von Willebrand factor content. Activated platelets produce TXA, and contact proteins to further enhance platelet aggregation via CAMP dependent receptor operated (ROC) calcium channels. They also secrete ADP and ATP. ADP recruits additional platelets while ATP and ADP degraded by endothelial cells into adenosine, inhibit platelets via A, receptor. ADP and ATP activate PzY receptors on the surface of endothelial cells inducing PGI, and EDRF (NO) release. Endothelin induces the contraction and proliferation of SMC. (Modified according to Boeynaems and Pearson [12].) B: Regulation of fibrinolysis at the endothelial surface. Plasminogen has high affinity for fibrin when activated by t-PA on endothelial surface. T-PA bound to endothelial cells remains active and protected from inhibition by plasminogen activator inhibitor 1 (PAI-1). (Modified according to Grulich-Henn and Miiller-Berghaus [21].)

112 duced by endothelial cells and vascular SMC and also prevents platelet aggregation, induces fibrinolysis and inhibits SMC proliferation by stimulating adenylate cyclase in platelets and SMC [7,8]. Endothelium derived relaxing factor (EDRF) which is a nitrosylated compound of arginine released from endothelial cells [9] is also responsible for vasodilation of SMC [lo]. EDRF stimulates soluble guanylate cyclase in platelets and increases cGMP which leads to an inhibition of platelet activation and aggregation. There is a close quantitative relationship between levels of intracellular Ca2+ and of PGI, and EDRF release [ll]. There is a synchronized reaction for PGI, and EDRF secretion during the interaction of platelets with the injured vessel wall [12] mediated by purinergic receptors (Fig. 2A). While PGI, and EDRF cause relaxation, the recently discovered endothelium derived constricting factor (EDCF) named endothelin-1 may contribute to vasoconstriction and SMC proliferation mediated by Ca2+ influx [13,14]. The transfer of lysophosphatidylcholine (lyso-PC) from oxidized LDL to endothelial membranes during the modification of LDL also produces a selective unresponsiveness to receptor regulated endothelium dependent vasorelaxation via a G protein mediated mechanism [15]. In addition, inflammatory mononuclear phagocytes entering the vessel wall inhibit prostacyclin synthetase by free radicals released during migration. Macrophages may also influence coagulation directly by secreting coagulation factors or indirectly by cytokines (tumor necrosis factor (TNF), interleukin 1 (ILl)), which induce the expression of tissue factors on endothelial cells [16,17]. In response to stimulation by IL-l, TNF, y-interferon (IFN-7) or lipopolysaccharide endothelial cells, like macrophages express class II major histocompatibility antigens (MHC II) on their surfaces, which can promote antigen presentation, binding and activation of T helper and cytotoxic T lymphocytes [18]. Activated endothelial cells express adhesion molecules for leukocytes, Fc receptors and secrete IL-l. In addition, their arachidonic acid pathway and respiratory burst is induced pathologically, and this may contribute to further injury of the endothelial cells [19], and activates the

complement, coagulation and fibrinolytic cascade. Endothelial cells may promote or inhibit fibrinolysis depending on their state of activation. The role of endothelial cells in the regulation of fibrinolysis is summarized in Fig. 2B. The vascular endothelium is thought to be the main source of tissue plasminogen activator (t-PA), the enzyme which is responsible for conversion of plasminogen to plasmin. It activates plasminogen efficiently only when it is bound to fibrin on the endothelial surface. The production and release of t-PA has been suggested to be modified by CAMP [20]. PAI- is also synthesized and secreted by endothelial cells when endothelial cell injury occurs to inhibit fibrinolysis [21]. Both in hypertension and in arterial diseases t-PA and PGI, secretion is decreased while the level of PAI- is increased or unchanged. Tissue reactions during fibrinolysis produce fibrin degradation products which in turn induce superoxide anion production from endothelial cells [22]. The high concentration of fibrinogen in atherosclerosis-related intimal lesions suggests that there is binding of fibrinogen to LDL within the intima, while Lp(a) with its homology to plasminogen binds to the same binding site as plasminogen and inhibits fibrinolysis providing a combined effect in promoting atherosclerosis [23]. Based on these observations, endothelial cells seem to possess a wide range of functional adaptability to respond to the hemostatic stresses of injury and inflammation, which in turn induces pathological events within the vessel wall. Effects of Ca’+ channel blockers on endothelial integrity, thrombosis and fibrinolysis

The endothelial integrity is of major importance for the maintainance of the physiologic functions of the vessel wall. It has been demonstrated that Ca2+ channel blockers exert protective effects on the endothelium (Table 1). VanHoutte [35] reported that Ca2+ channel blockers reduce the desquamation of endothelial cells triggered by a number of stimuli. Tedgui et al. [33] observed that nicardipine inhibits the permeation of albumin across the endothelial barrier induced by high potassium concentrations and StrohSchneider et al. 1341showed that nimodipine and

113 flunarizine inhibit the increased permeability observed in atherosclerotic plaques. Platelet activation is a critical parameter in atherosclerosis and is induced by Ca2+ either by inward Ca2+ flux across the cell membrane and/or by release from special storage organelles within the platelet [43,44]. It has been shown by different research groups that various types of Ca*+ channel blockers inhibit platelet aggregation and platelet secretion [45]. Ca*+ channel blockers exert an enhanced fibrinolytic potential by increasing t-PA synthesis [38,39]. These effects are mediated by receptor operated Cal+ channels and are summarized in Table 1. Although the precise mechanism of Ca’+ channel blocker action on platelet aggregability, endothelial cell adhesion and fibrinolysis has to be further investigated, the mentioned effects might

TABLE

tend to reduce the thrombo-embolic diovascular disorders. Role of mononuclear

phagocytes

risk in car-

in atherosclero-

sis

Another early key event in atherosclerosis is the enhanced chemotaxis, adhesion and infiltration of monocyte-derived macrophages to the subendothelial space [4,5]. Modification of LDL may play an important role in the initiation of monocyte chemotaxis. Endothelial cells are able to modify LDL through oxidation at their lipid and protein moieties [46]. The oxidatively modified LDL appear to possess a phospholipase A2 (PL A,) activity, a phenomenon that explains the hydrolysis of phospholipids (generation of lysoPC) in the lipoprotein 1471(Fig. 3A). Lyso-PC is

1

EFFECTS OF Caz’ FIBRINOLYSIS

CHANNEL

BLOCKERS

ON MEMBRANE

INTEGRITY,

VASCULAR

TONUS,

THROMBOSIS

Cell type

Effect

Drug type

Reference

Platelets

inhibit aggregation

nifedipine diltiazem diltiazem verapamil nifedipine nifedipine, verapamil nifedipine nicardipine nifedipine nifedipine

24 25 26 21 28 29

inhibit TXA,

release

decrease angiotensin inhibit phospholipase inhibit PDGF-release Endothelial

cells

cell integrity inhibit endothelial

II content activity

permeability

inhibit desquamation vascular tonus increase PGI, release thrombosis and fibrinolysis enhance fibrinolysis decrease P-TG and platelet factor 4 increase of t-PA no change of PAI- secretion Smooth cells

muscle

prevent

Ca’+-overload

enhance PGl,-effects inhibit endothelin 1 induced

contraction

diltiazem,

30 31 37

nicardipine nimodipine. flunarizine nifedipine

33 34

nifedipine

3h

isradipine felodipine isradipine isradipine

37 38 39 39

nifedipine, diltiazem verapamil verapamil, diltiazem nimodipine

40

35

41 42

AND

114 generated at the surface of the LDL particle and transIocated to the endothehal cell membrane [15]. Lyso-PC can induce the chemotactic attraction of monocytes to the subendothelial space where it subsequently inhibits the motility of macrophages 1481.Evidence for the in vivo modification of LDL comes from the findings that autoantibodies against oxidized LDL can be demonstrated in human sera [49]. These LDL immunocomplexes are taken up via an Fc receptor mediated process by macrophages [50]. Monocytes which have migrated into the subendothelium differentiate into macrophages and lose their ability to bind normal LDL but

w

ingest large amounts of modified LDL via the scavenger receptor pathway 1511. According to recent reports, different scavenger receptor binding sites exist, to which modified LDL can bind [52]. The physiological significance of the scavenger receptors is widely accepted. It is considered that oxidized LDL particles and especially the released fatty acids are cytotoxic for endothelial cells and induce endothelial cell injury and stimulate platelet aggregation [53]. However, the increased removal of these cytotoxic particles results in foam cell formation with enhanced cholesteryl ester content [54]. The stored cholesteryl esters undergo a continuous cycle of hydrol-

circulating Monocytes native LDL

resident Monocyte/Macrophages

oxidized LDL

Media Fig. 3. A: Initiation of atherogenesis by oxidizied LDL. LDL can be oxidatively modified by endothelial cells, macrophages and smooth muscle cells. Oxidized LDL with its cytotoxic effect injures endothelial cells and stimulates cell proliferation. The modified LDL is taken up via the scavenger receptor pathway by macrophages which turn into foam cells. Lyso-PC generated in oxidized LDL particle by PL A,, is chemotactic for monocytes but inhibits migration of intimal macrophages. (Modified according to Steinberg et al. [50].) B: Stimulation of PDGF secretion by oxidized LDL. Oxidatively modified LDL is able to induce PDGF(BB) secretion from macrophages and PDGF(AA) secretion from smooth muscle cells. This latter is further enhanced in an autocrine manner. Stimulated endothelial cells can also secrete growth factors. During platelet aggregation induced by endothelial injury, platelets are the main sources of PDGF(AB) secretion.

115 native LDL

0

Endothellal injUrY

< Endothelial cell

Fig. 3 (continued).

ysis and re-esterification [55] by acid and neutral cholesteryl ester hydrolases (ACEH, NCEH) and acyl CoA : cholesterol acyltransferase (ACAT) until cholesterol is used for membranes and secretion. An effective cholesterol efflux from macrophages is critical for the prevention of foam cell formation and is promoted by extracellular cholesterol acceptors such as HDL. Macrophages secrete lipoprotein lipase which, upon metabolic overload, can facilitate the uptake of metabolic products originating from triglyceride rich lipoproteins such as P-VLDL or hypertriglyceridemic-VLDL, (HTG-VLDL ,> occurring in certain forms of dyslipoproteinemias [56,57]. The behaviour of the lipid ingesting macrophages can be altered by inflammatory activation due to the multilateral interactions in the

vessel wall. Immune activation of macrophages has been reported to cause changes in their lipid and lipoprotein metabolism [58-601. Activated macrophages may produce many of the activating factors themselves, such as IL-l, TNF, GM-CSF, complements, PDGF, transforming-growth factor p (TGF-P), prostaglandins and leukotrienes, indicating that macrophage activation may be amplified or decreased by autocrine regulatory loops [61]. On the basis of the above described phenomena migration of monocytes, their differentiation into macrophages and their transformation into foam cells or activated cells are the critical events during early lesion development. Immune activation is also modulated by atherogenic lipoproteins [62], and thus, besides cholesterol overloading of the cells, lipoproteins can also

116 affect cellular activation, differentiation liferation.

and pro-

Role of Ca’+ channel blockers on chemotaxis and migration of neutrophils, monocyte /macrophages Ca2+ channel blockers are able to modulate chemotaxis of blood monocytes and lipid metabolism in monocyte derived macrophages (Table 2). It has been demonstrated that nisoldipine reduces formyl-methionyl-leucyl-phenylalanine (FMLP) mediated Ca2+ flux and that verapamil decreases chemotaxis in neutrophils [65,661. In general, Ca2 + channel blockers may inhibit chemotactic migration by decreasing receptor mediated Ca2+ flux thus leading to a reduced arachidonic acid metabolism. These migration processes involved in atherosclerosis are mediated by a coordinated interaction of integrins with the cytoskeleton. Several integrins can localize near focal contacts, where the cell membrane is closely opposed to the extracellular matrix and where actin bundles terminate [67]. Actin assembly and dissembly are regulated by products of lipid hydrolysis via PL A, activity (arachidonic acid) or one of their metabolites (platelet activating factor (PAF), leukotriene B, (LTB,)). Ca2+ channel blockers may inhibit chemotaxis by interfering with these mechanisms. Role of Ca’+ channel blockers on cellular lipid metabolism The chemotactic attractance of monocyte/macrophages leads to their accumulation in the

TABLE

2

EFFECTS

OF Ca*+

CHANNEL

BLOCKERS

ON CHEMOTAXIS

FMLP = formyl-methionyl-leucyl-phenylalanine; Cell type Smooth cells

subendothelial space, where they are capable of ingesting huge amounts of lipoproteins, an effect which is modified by CaZf channel blockers. We demonstrated that cholesterol efflux is mediated by two major routes and influenced by nifedipine [68] (Fig. 5). The first pathway is related to an HDL receptor dependent release, in which apo A-I-rich HDL bind specifically to an HDL-binding protein on macrophages. These apo A-I-rich HDL particles are internalized into a non-lysosomal compartment and take up cholesterol from lamellar bodies which are formed from cytoplasmic lipid droplets upon attachment of endoplasmic reticulum or are released from the trans-Golgi network. The second route is related to an HDL-receptor independent release of cholesterol by formation of lamellar bodies originating from lysosomes. These lamellar bodies move towards the cell periphery, attach to the cell membrane and release their lipid components into the extracellular medium or into the cell membrane. This mechanism of cholesterol release is promoted by apo A-I/A-IV/cholesteryl ester transfer protein (CETP)/ lecithin : cholesterol acyltransferase (LCAT)-rich HDL particles which preferentially bind non-specifically to the cell membrane. This cholesterol transfer and the generation of these lamellar bodies is promoted by Ca2+ channel blockers such as nifedipine, which downregulate HDL-binding and enhance sphingomyelin @PM)-synthesis in macrophages (Table 3). In additon, Ranganathan et al. [781 demonstrated in skin fibroblasts that verapamil and diltiazem decrease LDL-degradation due to their lysosomotropic properties. This is in accordance with the observation of Stein et al. [691 that verapamil enhances LDL-binding and uptake, but

muscle

Neutrophils

12-HETE

= 12-L-hydroxy-5,8,10,14-eicosatetraenoic

acid.

Effect

Drug type

Reference

inhibit 12-HETE induced chemotaxis inhibit IL-l, LTB,, PDGF mediated migration

nicardipine nivaldipine

63 64

inhibit chemotaxis inhibit FMLP mediated

verapamil nisoldipine

65 66

Ca2+-flux

117

YY

AcLDL scavenger

pathway

I

secretory vesicle

coated vesicle

’

I lysosome

0

\

A-IV/LCAT-HDL

Nifedipine bodies

ACAT 1

lipid

/-

-

A-I-HDL

droplet

Fig. 4. Two major routes of cholesterol release in macrophages. (1) Upon cholesterol loading via scavenger-receptor macrophages generate cholesterol- phospholipid containing lamellar bodies which originate from lysosomes. This pathway is promoted by Nifedipine (see text). (2) HDL-receptor mediated cholesterol efflux, in which apo A-I-rich HDL bind specifically to a 110 kDa protein. These apo A-I-rich HDL pariticles are internalized into a non-lysosomal compartiment and take up cholesterol from lamellar bodies, which are formed from cytoplasmic lipid droplets upon attachment of endoplasmatic reticulum or are released from trans-Golgi network. ACAT _L = acyl CoA: cholesterol acyltransferase inhibitors.

also causes a delay in lysosomal degradation of LDL. Paoletti et al. [Sl] reported that verapamil and diltiazem stimulate LDL uptake, while LDL-degradation was slightly reduced. Additionally, Ca2+ channel blockers affect the hydrolytic enzyme activities involved in cholesterol metabolism. Etingin et al. [70] found that nifedipine increases ACEH and neutral cholesteryl ester hydrolase (NCEH) activity in vascular SMC, possibly induced by an enhanced intracellular CAMP level. Recently they reported that nifedipine and diltiazem increase cholesteryl ester hydrolysis and enhance CAMP-levels in the vessel wall [73].

Smooth muscle cell proliferation Another characteristic feature of atherosclerotic plaques is the migration of vascular SMC from the media into the intima where they are transformed from the contractile into a synthetic phenotype and proliferate induced by various cellular products [64]. A recent model of this process has been described by Thyberg et al. 161. Normally, the basement membrane helps to keep the SMC in a contractile phenotype [82]. When this barrier is damaged the plasma membrane of SMC interacts with macromolecules from

118 TABLE 3 EFFECTS OF Ca2+ CHANNEL BLOCKERS ON LIPID METABOLISM PL = phospholipids, TG = triglycerides, CE = cholesteryl ester, UC = unesterified cholesterol, ACEH = acid cholesteryl hydrolysis, NCEH = neutral cholesteryl ester hydrolysis, ACAT = acyl-CoA: cholesterol transferase. Cell type

Effect

Drug type

Reference

Smooth muscle cells

enhance LDL-binding, LDL-uptake and LDL-degradation increase ACEH, NCEH activity decrease PL, TG, CE content decrease cholesterol content

verapamil

69

nifedipine verapamil nifedipine, diltiazem, verapamil nifedipine, diltiazem

70 71 72

verapamil verapamil nifedipine verapamil

74 75

nifedipine

68

increase CAMP-level and CE hydrolysis Monocyte/macrophages

enhance LDL-binding, LDL-uptake inhibit ACAT activity inhibit delivery of UC from lysosome to ACAT promote lamellar body formation reduced lipid droplet generation

73

76

Fibroblasts

enhance LDL-binding, LDL-uptake reduced LDL-degradation enhance LDL-uptake and -degradation

verapamil verapamil verapamil

77 78 19

J 714

decrease ACAT-activity

verapamil

80

MDM

increase LDL-uptake and -degradation

verapamil

74

the blood or produced locally by cells in the vessel wall [82]. Among them fibronectin binding to adhesion receptors seems to play a major role in the transition of SMC from contractile to a synthetic phenotype 1831, when there is a switch from SMC specific a-actin to p-actin and a decrease in the expression of SMC myosin 1841.The modulation into a synthetic phenotype is associated with increased binding and degradation of VLDL and LDL [85]. After the phenotypic modulation SMC proliferate in response to polypeptide mitogens, vasoactive hormones, prostaglandins and leukotrienes. Among them PDGF is the most potent mitogen for vascular SMC proliferation so far tested and also a chemoattractant [861. It is a 30 kDa basic protein consisting of two disulphide linked polypeptide chains A or B that give rise to three disulfide-linked dimers (AA, AB, BB) [87]. PDGF, similar to other growth factors exerts its effects through specific cell surface membrane receptors in accordance to the A or B chains. The two PDGF-receptors (a, /3) are present in differ-

ester

ent concentrations on various cell types. The (Yreceptor binds either the A or the B chain, while the P-receptor binds only the B chain [87]. Therefore the capacity of the different isoforms of PDGF to ‘induce mitogenesis depends on the specific PDGF isoform and the relative numbers of receptor subunits present on the corresponding cell. Both types of PDGF receptors are autophosphorylated by a tyrosine kinase and inositol-1,4,5triphosphate (IP,) turnover, intracellular Ca2+ release is induced after activation [88]. However, PDGF-BB has a greater efficiency than PDGF-AA on IP, release, on the formation of 1,2-diacylglycerol (DAG) and on Ca2+ mobilization, w’hich is also associated with vasoconstrictor activity and effective mitogenicity. On the other hand PDGF-AA binding to its receptor on the surface of vascular SMC is more potent than PDGF-BB in stimulating protein kinase C [32]. The regulation of the growth factor receptors is supposed to be mediated by a group of GTP binding proteins which are likely to belong to a

119

family of farnesylated proteins. The farnesylation of these proteins occurs during the mevalonate pathway probably providing a linkage between cellular cholesterol metabolism and proliferation ]891. During the phenotypic transition SMC start to express PDGF cy- and P-receptors to respond to all isoforms of PDGF released cell types during endothelial injury and synthesize mRNA for the A chain of PDGF [903. The secretion of PDGF by SMC themselves therefore serves as a selfamplifier mechanism in an autocrine regulatory loop to further enhance the proliferative response (Fig. 3B). Cytokines such as IL-l, IL-6 and IFN-y released during macrophage-T cell interaction also have the ability to modulate PDGF gene expression in various cells of the vessel wall [91,92]. Other growth factors may be of similar importance in atherogenesis which has been reviewed by Thyberg et al. [6]. Vasoactive hormones (vasopressin, norepinephrin) which are potent vasoconstrictors in vivo stimulate proliferation of SMC via adrenergic receptors. Moreover, growth factors can induce contraction of aortic slices [93] and the mitogenic vasoconstrictor peptide endothelin is also secreted from SMC after stimulation with hormones and growth factors [14]. Products of arachidonic acid metabolism also exert effects on the proliferative response of SMC 1941. Leukotrienes promote proliferation [95], while prostaglandin E, (PGE,) and prostaglandin E, (PGE,) inhibit the induction of DNA synthesis by PDGF [951. There is an opposing effect between PGI, and PDGF. PDGF can induce PGI, secretion and by a feed back regulation can inhibit PDGF release [96]. Adenosine and adenosine nucleotides which are responsible for vascular endothelium and platelet aggregation, have also been found to inhibit the mitogenic effect of PDGF on SMC [97]. There is a close relationship between the inhibitory effect of these substances and their ability to raise the intracellular concentration of CAMP in SMC [981. Furthermore several plasma proteins including a,-macroglobulin have the ability to interact with PDGF and inhibit its binding to the PDGF receptor [99]. Therefore, the final rate of SMC proliferation, and consequently lesion progression, is obviously

dependent on the balance between growth-promoting and growth-inhibiting substances released during cellular interactions within the vessel wall. Effects of Ca2+ channel blockers on smooth muscle cell proliferation Cell proliferation is an essential factor in atherosclerosis and can be modulated by Ca*+ sensitive processes. There is growing evidence that Ca*+ channel blockers inhibit SMC proliferation, which might explain part of their therapeutic value in atherosclerosis (Table 4). Stein et al. [105] have shown that verapamil decreases SMC proliferation by inhibiting DNA synthesis. It has been shown that nifedipine interrupts the linkage between the mitogenic trigger PDGF and the proliferative response of the synthetic phenotype of SMC. PDGF stimulates phosphoinositol turnover leading to a mitogenic and vasoconstrictive response accompanied by an increased formation of IP,, DAG and a rise in intracellular Ca’+ levels and activation of protein kinase C. Block et al. [321 reported that these processes are inhibited by nifedipine. Alternatively, these effects could also be mediated by CAMP dependent mechanisms. Cheung et al. [ll l] showed that

TABLE

4

EFFECTS OF Ca” PROLIFERATION

CHANNEL

BLOCKERS

ON

Cell type

Effect

Drug type

Reference

Platelets

inhibit PDGF release

nifedipine

32

Smooth muscle cells

inhibit cell proliferation

nifedipine flunarizine isradipine nifedipine verapamil nivaldipine verapamil isradipine nimodipine nifedipine nitrendipine nifedipine nifedipine

inhibit phosphoinositol-turnover

10 101 102 103 71 104 10s 106 107 108 109 110 32

SMC

nifedipine and Bay K 8644 inhibit the binding of adenosine to its A, receptor on SMC leading to an increased CAMP level which decreases PDGF-induced DNA synthesis. In summary, the present data strongly indicate that CaZf channel blockers impair the development and progression of atherosclerotic plaque formation by reducing vascular SMC proliferation and might also influence the transformation of the contractile to the synthetic phenotype of SMC. However, the specific mechanism by which second messengers (CAMP, Ca2+) are involved in these processes has to be further elucidated. Extracellular martix and cell adhesion A hallmark in the progression of atherosclerosis is the accumulation of extracellular matrix components in the intimal layer, but it remains to be determined why this accumulation occurs. Extracellular matrix components modulate several cellular processes like adhesion, migration and proliferation. Collagen as a structural molecule assembles into a meshwork that forms the core of all basal laminae. It binds to many molecules

including oxidized LDL, which is deposited in collagen-rich tissues such as tendons and atherosclerotic plaques. The scavenger receptor might compete with collagen for binding of oxidized LDL 1511.Elastin degradation products are capable of stimulating oxidative burst and are chemotatic for monocytes and fibroblasts [ 1121. Fibronectin as an adhesive glycoprotein is involved in cell attachment, spreading, and organization of actin cytoskeleton and promotes the conversion of rat aortic SMC from a contractile to a synthetic phenotype. Increased amounts of fibronectin are found in early atherosclerotic lesions [113]. Laminin which is found mainly in the basal laminae is connected with collagen type IV and proteoglycans, and might counteract these fibronectin effects. Both proteins are interwoven in a hydrated gel composed of a network of glycosaminoglycan chains.The glycosaminoglycans are covalently linked to proteins to form proteoglycans. Proteoglycans regulate the binding of enzymes (e.g., lipoprotein lipase (LPL)), growth factors (e.g., ECGF), coagulation (thrombin) and anticoagulation factors (antithrombin): They are involved in cell adhesion and cell-cell association

native LDL

0

ECGF

Thromhin Antilhrombin

m

#

Endothelial

cell

collagen fibrillogenesis migration and PrOiiferatiOn

Fig. 5. Extracellular matrix components involved in atherosclerosis. Glycosaminoglycans have binding sites for proteins such as endothelial derived growth factor (ECGF), platelet factor 4 (PF 4) or lipoproteinlipase (LPL), maintain viscoelastic properties and modify LDL to oxidized LDL (oxLDL), which is ingested by macrophages. (Modified according to Wight [114].)

121

processes, migration and proliferation of SMC and collagen fibrillogenesis (Fig. 4). They are subdivided into 3 chemical classes [1141 with different functions: Dermatan sulfate proteoglycan (DSPG) is involved in the assembly of fibrils and collagen structures; heparan sulfate proteoglycan (HSPG) physiologically inhibits SMC proliferation, an effect which is possibly disturbed in atherosclerosis leading to enhanced HSPG-degradation. However, the precise molecular mechanism has still to be elucidated. It is hypothesized that these glycosaminoglycans are internalized by the cells and directly interfere with the cell nucleus thus inhibiting cell proliferation. Furthermore, the permeability of the basal membrane is increased, allowing facilitated migration of SMC into the intima. Chondroitin sulfate proteoglycan (CSPG) synthesis is enhanced by LDL. Camejo et al. [115] demonstrated that incubation of LDL with arterial CSPG increases LDL uptake by monocytederived macrophages in man, indicating that proteoglycan is capable to modify LDL. These findings suggest that charge alterations of LDL molecules influence their recognition by macrophages leading to internalization via the scavenger pathway.

Effects of Ca’+ channel blockers on extracellular matrix and adhesion processes Ca2+ channel blockers may stabilize the integrity of the vessel wall by modulating adhesion processes and they are capable of inhibiting enhanced extracellular matrix synthesis (Table 5). Dietrich et al. [116] observed that verapamil inhibits collagen synthesis in fetal rat bone. Orekhov et al. [71] reported that verapamil diminished the [ “Hjproline incorporation into collagen. However, it has to be mentioned that high drug concentrations were administered which might be cytotoxic. Weinstein et al. [117] found that collagen synthesis is reduced by isradipine in vascular SMC, skin fibroblasts and macrophages. These results indicate that this effect is independent of Ca*+ flux across voltage operated Ca2+ channels, because fibroblasts and macrophages do not posses this type of Ca2+ channel (see Table 5). Effects of Ca’+ channel blockers on cell membrane integrity Cell membranes consist of a continuous bilayer of lipids in which various membrane proteins are embedded. The lipid portion, containing phos-

TABLE 5 EFFECTS OF Ca*+ CHANNEL BLOCKERS ON STRUCTURAL

ELEMENTS INVOLVED IN ATHEROSCLEROSIS

Structural elements

Effect

Drug type

Reference

Extracellular matrix

inhibit collagen synthesis

verapamil

116

verapamil isradipine

71 117

nifedipine, diltiazem nifedipine nifedipine, verapamil nifedipine nifedipine, diltiazem verapamil nifedipine, diltiazem verapamil, isradipine diltiazem, verapamil nifedipine, verapamil nifedipine, diltiazem verapamil

118 119 120 121 119

Cell membrane

induce membrane perturbations scavenge superoxide anions scavenge free radicals accumulate in cell membrane inhibit malondialdehyde formation

inhibit lipid peroxidation

122 123 120 124

122 pholipids, cholesterol and glycolipids, determines the basic structure of the membrane and the proteins are responsible for most membrane functions like signal transduction, enzyme regulation or molecule transport. The plasma membrane represents a selective filter and so it is of major interest how Ca 2+ channel blockers interfere with cell membranes and change cellular processes. In general, Ca2+ channel blockers are hydrophobic molecules, which can be differentiated according to their solubility properties [125]. Ca2+ channel blockers of the verapamil and diltiazem group are positively charged in solution, while the dihydropyridines (e.g., nifedipine and nitrendipine) are uncharged and accumulate in plasma membranes and lipoproteins. Herbette et al. [121] reported that dihydropyridines first interfere unspecifically with the lipid bilayer and then bind to specific receptor binding sites upon lateral movement. Shi et al. [1181 observed that verapamil and diltiazem cause large perturbations in membrane bilayer structures due to their electrical properties. Our own experiments have shown that Ca2+ channel modulators significantly change cellular phospholipid metabolism [ 1261. The final step in SPM synthesis which takes place in the cis/ tram Golgi region obviously is a limiting factor in membrane formation and it is increased by Ca*+ channel blockers while phosphatidylcholine synthesis is not affected. In the cell membrane SPM is closely associated with unesterified cholesterol and anchors it in the outer membrane leaflet and Ca*+ channel blockers might attach more unesterified cholesterol due to their ability to enhance SPM synthesis. Thus HDL is capable of taking up more unesterified cholesterol from peripheral cell membranes and thereby may improve reverse cholesterol transport. In addition to phospholipid synthesis obviously also phospholipid catabolism plays a major role in atherosclerosis. Slotte et al. [127] demonstrated that SPM strongly influences the distribution of unesterified cholesterol. They could show that an increased degradation of cell membrane SPM by SPMase leads to a reduced translocation of unesterified cholesterol to the membrane surface and thereby promotes cholesterol esterification by the microsomal enzyme ACAT. In this context various physiological PL

A, activities are also of importance. The HDL associated enzyme LCAT hydrolyses PC to lysoPC and the released fatty acid is transferred to cholesterol for esterification. Kugiyama et al. [18] described a similar PL A, activity in LDL particles. Modulation of signal transducers by Ca2+ channel blockers - role of Ca2+ in regulatory processes of vessel wall cells The question arises as to which molecular mechanisms induce the antiatherogenic effects of Ca2+ channel blockers. The data suggest that these drugs exert their effects by multiple mechanisms: on the one hand these effects can be mediated by reducing intracellular Ca2+ levels, on the other hand by interfering with membrane proteins. In addition, some effects are based on the lysosomotropic properties of verapamil and diltiazem. In the early lesion Ca2+ mainly influences atherogenesis by changing metabolic processes induced by modulation of Ca2+ influx pathways and signal transducers, whereas in the advanced lesion CaZf overload is the characteristic feature. In this context it is necessary to look at the physiological role of Ca2+ during the early lesion development [128]. Normally, Ca 2f have only a sm all capacity to permeate cell membranes, and Cazf have to be pumped out of the cell against a concentration gradient. The cell is highly sensitive to changes of intracellular Ca 2+ levels and these changes are caused by certain ligands which upon binding to specific cell membrane structures regulate the permeability for Ca*+. Intracellular Ca*+ controls several physiological processes like muscle contraction, cell adhesion, differentiation and growth processes, secretion of neurotransmitters, hormones and metabolic enzymes, transport of minerals and nutrients. Ca2+ binds with a high specificity to various proteins thus changing their conformational structure leading to activation of these proteins. The most important Ca*+ binding protein is calmodulin, which modulates numerous functions mentioned above. The intracellular Ca2+ concentration is physiologically regulated by interfering feedback mechanisms. If these regulatory processes are disturbed multiple metabolic

123 dysregulations may be the result of the aberrant Ca*+ concentrations. In early lesions the formation of fatty streaks has been attributed partially to increased intracellular Cazf levels [40]. Ca2+ is essential for LDL binding to its receptor, increases endothelial permeability, induces chemotaxis of macrophages and neutrophils, stimulates oxidative burst and is involved in the regulation of proliferative processes. Therefore this is a sensitive target for antiatherogenic therapy. Various drug classes, such as Ca2+ channel blockers, interfere with these processes in different ways. It is the target of antiatherosclerotic therapy to reduce the still reversible generation of fatty streaks and to thereby inhibit the progression of atherosclerosis (Fig. 6).

Interaction of Ca ‘+ channel blockers with voltage operated Ca*+ channels An increased Ca *+ flux through cell membranes can be regulated by voltage operated Ca*+ channels. This can either be achieved by depolarisation of plasma membranes or by binding of Ca* + channel agonists (e.g., Bay K 8644) to the dihydropyridine receptor [40]. Recently, it has been shown that the dihydropyridine receptor consists of five subunits and is possibly regulated by CAMP dependent phosphorylation [129]. Ca2+ channel blockers can inhibit the Ca*+ flux across the plasma membrane in cells with voltage operated Ca2+ channels. However, among the cells involved in the atherosclerotic process (endo-

Extracellular signal

t

1Signal transduction /

1

Voltayyated

/

G-Protein mediated

+

I

1

Phosphorylation

4

]

(

Cellfunction

J

J

4 Gene expression

Fig. 6. Different mechanisms of Ca *+-flux across the plasma membrane modulating cellular functions. Ca*+ channel blockers bind to the ru,-subunit (1.50-170 kDa) of voltage operated Ca *+ channels and inhibit transmembrane Ca ‘+-flux. The p-subunit (54 kDa) is probably concerned with the nucleoside transporter. Transmembrane Ca 2t-fl~~ can also be induced by the activity of certain G proteins. The GCa2+ protein directly opens Ca2+ channels, whereas the G, protein activates the hydrolysis of PIP, leading to transient Ca* + flux from intracellular storages.

124 thelial cells, monocyte/macrophages, T-lymphocytes, smooth muscle cells, platelets) only SMC express voltage operated Ca*+ channels [130]. There is evidence that in the other cell types different mechanisms, such as receptor operated or G protein operated Ca*+ channels, might play a role.

directly opens Ca*+ channels, whereas the G, protein activates the phosphoinositol specific phospholipase C (PI-PLC), which stimulates the hydrolysis of phosphoinositol-4,5-bisphosphate (PIP,) to IP, and DAG [1321. IP, is released into the cytosol and induces a transient Ca*+ flux from intracellular storages. Calmodulin binds the released Ca*+ and a series of proteins is activated by phosphorylation dependent processes. The transient Ca*+ increase stimulates proteinkinase C, which phosphorylates another group of proteins. The resulting sustained Ca*+ influx possibly occurs by gating of Ca*+ channels. The Ca*+ influx, however, is not sufficient to trigger a cellular response; there are additional membrane associated transducers necessary to convert this second messenger signal in the respective cell types. The known therapeutic side effects of Ca*+

Interaction of receptor operated and G protein mediated Ca2+ channels with Ca2+ channel blockers The precise role of signal transducers which mediate Ca*+ flux has not yet been elaborated [1311. According to Birnbaumer et al. [132] an agonist/receptor binding is assumed to modulate the activity of certain G proteins which alter gating of Ca *+ channels [133]. The Gcaz+ protein

Swarlaml~ cl

PI Ecto-ATPases

Ca++ channel blocker ATP > AMP > adenoslne

adenoslne > AMP > ATP

I

a.“-m4TP > 2 h4es.ATP

contraction A;P

i WC acid

RiA DNA

. . Proteinkinase

mm

[

Phosphorylation

C

/d

w cellular function Fig. 7. The superfamily of purinergic receptors are divided into various subtypes which modulate the second messenger system (CAMP Ca*+). Ca2+ channel blockers of the dihydropyridine (DHP) type inhibit the binding of adenosine to AI-receptor leading binds with high affinity to the nucleoside to an increase of the intracellular CAMP level. The Ca *+ channel blocker (+)-nimodipine transporter which has structural homology to the p-subunit of the DHP receptor.

125 channel blockers have indicated that these drugs also modulate other signal transducers e.g. purinergic receptors [ 1111, the nucleoside transporter [ 1341, peripheral benzodiazepine receptors [135] and the multi-drug resistance protein [1361. This may lead directly or indirectly to significant influences on the intracellular Ca*+ concentration or to other second messenger related effects. Interaction purinergic porter

channel blockers with of Ca’+ receptors and the nucleoside trans-

Burnstock [137] divided the purinergic receptors into two classes as shown in Fig. 7. P, receptors are more sensitive to adenosine than to any of its nucleotides and P, receptors having a converse selectivity, favoring binding affinities of ATP > AMP > adenosine. P, receptors are supposed to act via an activation or deactivation of adenylate cyclase while P, receptors act as far as is known by the phosphoinositol cycle pathway and mobilization of Ca2+ from intracellular stores. From a structure-activity perspective the Pi receptors are subdivided into A, and A, receptors. The A, receptor is responsible for inhibition of adenylate cyclase by adenosine at nanomolar concentrations. The A, receptor appears to have low and high affinity subtypes for adenosine and activates adenylate cyclase at micromolar concentrations. Dihydropyridines have been shown to inhibit high affinity binding of adenosine to A, receptors [ Ill] and thereby enhance the intracellular CAMP level. CAMP regulates numerous important functions such as the key enzymes of cholesterol homeostasis (ACEH, TABLE EFFECTS

NCEH) or cytoskeletal elements involved in endocytosis and secretion processes. Endothelial P, receptors influence the interaction between platelets and the endothelial layer of the arterial wall as shown in Fig. 2A. During ischemia and hypoxia, platelets rapidly release dense granules containing ATP and ADP and other factors. ATP and ADP induce platelet aggregation via P, purinergic receptors. However, ATP is degraded to adenosine by ectonucleotidases which inhibit platelet aggregation [12]. Adenosine acts on platelets via A, receptors thus increasing intracellular CAMP levels of platelets. The enhanced CAMP level is a possible explanation of the antiaggregatory properties of Ca*’ channel blockers. They may modulate the PGI,/TXB, balance resulting in an enhanced release of PGI,, which inhibits platelet aggregation and relaxes smooth muscle cells (Table 6). On the other hand, adenosine can be taken up by the nucleoside transporter, a membrane associated 54 kDa protein, which regulates adenosine transport through cell membranes. This protein has structural homology to the P-subunit of the dihydropyridine receptor. It is important to note that enantiomers of dihydropyridines and phenylalkylamines generally have opposite stereoselectivity for the nucleoside transporter and voltage operated Ca*+ channels. Striessnig et al. [134] observed that (+ I-nimodipine binds with an affinity more than tenfold higher than (-- I-nimodipine to the nucleoside transporter, whereas ( - )-nimodipine binds to voltage operated Ca*+ channels with an affinity 5-fold higher than its (+ l-enantiomer. These data suggest that Ca *+ channel blockers do have specific binding sites on voltage operated Ca*+ chan-

6 OF PURINERGIC

RECEPTORS

ON CELLS

INVOLVED

IN ATHEROSCLEROSIS

Cell type

Effect

Receptor

Platelets Endothelial ceils Smooth muscle cells

antithrombosis vasodilation vasodilation, relaxation vasoconstriction vasodilation inhibition of oxidative burst suppression of phagocytosis inhibition of growth and cell toxicity

A: A,

Neutrophils Macrophages T lymphocytes

A2

P zx P zy A? AZ A?

type

Reference 138 139 I40 141 141 142 143 144

126 nels and the nucleoside transporter, but that these binding sites have different stereoselectivity. Interaction of Ca*+ channel blockers ripheral benzodiazepine receptors

with pe-

The physiological functions of peripheral benzodiazepine receptors are not yet precisely known [145]. However, they are involved in cellular processes such as differentiation and proliferation. Peripheral benzodiazepine receptors are located in the outer membrane of mitochondria, the endoplasmic reticulum and plasma membranes of several cell types (monocytes, platelets, SMC) [146]. Endogenous ligands are porphyrins which play a role as prosthetic groups in the formation and translocation of cytosolic proteins such as hemoglobin and catalase. In addition, two proteins, which might play an important role in atherosclerosis, are known to interact with peripheral benzodiazepine receptors. The first protein is diazepam binding inhibitor (DBI)/endozepine which is identical with acyl CoA binding protein (ACBP) and binds with micromolar affinity to peripheral benzodiazepine receptors. Our experiments showed that ACBP/DBI inhibits ACAT mediated cytoplasmatic cholesterol esterification (Schmitz, G., Beuck, M., Kerkhoff, C., Triege-Rasmussen, J., Spener, F. and Knudsen, J., unpublished data). Other data show that ACBP/DBI promotes cholesterol uptake into mitochondria [147]. The second protein with a size of 16 kDa associated with an endogenous PL A, activity binds to peripheral benzodiazepine receptors and dihydropyridine receptors with a micromolar affinity [148]. It is not yet clear whether the 16 kDa protein interferes directly with the receptor or by released fatty acids. Beaumont et al. [149] demonstrated that phospholipids and unsaturated fatty acids inhibit the binding to peripheral benzodiazepine receptors. The general mechanism of peripheral benzodiazepine receptor regulation is not yet understood. However, it has been shown that the peripheral benzodiazepine receptors effects are not controlled by y-aminobutyric acid (GABA) mediated gating of Cl- channels. Rampe et al. [150] hypothesized a model to explain the complex interactions of Ca2+ channel blockers and benzodiazepines with pe-

ripheral benzodiazepine receptor and Ca2+ channels. The peripheral benzodiazepine antagonist PK 11195 stabilizes voltage operated Ca2+ channels in the resting state and blocks the binding of Ca*+ channel blockers (nifedipine, verapamil, diltiazem) and agonists (Bay K 8644) as well as benzodiazepines (diazepam, Ro 5-4864). Ca2+ channel blockers and benzodiazepines interfere with the closed Ca 2+ channel, whereas the Ca2+ agonist interferes with the opened state. Apparently peripheral benzodiazepines and Ca2+ channel blockers modulate Ca2+ channels as well as peripheral benzodiazepine receptors in a complex way. Irrteraction protein

with

multi-drug

resistance

&IDR)

The MDR protein P 170 is a transporter, which carries hydrophobic molecules such as cytostatic drugs out of the cell [136]. It is an ATP-binding molecule with characteristics of a pore forming protein. Various Ca2+ channel blockers (verapamil, diltiazem and possibly dihydropyridines) reverse this drug resistance by inhibiting the energy dependent transport of hydrophobic compounds. Interestingly, adenylate cyclase, MDR protein and the c-u,-subunit of voltage operated Ca2+ channels have similar transmembrane domains. This side effect of Ca2+ channel blockers might be attributed to similar binding sites [151]. Conclusion

Atherosclerosis is a multifactorial disease in which the impaired intracellular signal transduction seems to induce the disturbance of intercellular communications within the vessel wall. Ca2+ as an important second messenger is involved in certain regulatory processes in the vessel wall. Thus, it promotes LDL receptor binding, induces chemotaxis of monocyte/macrophages and smooth muscle cells, promotes the migration of these cells into the intima and stimulates the secretion of collagen and other components. This review emphasizes how Ca2+ channel blockers interfere with these mechanisms and their potential role in risk factor intervention. In addition to

127 the well characterized blockade of voltage operated Ca2+ channels they exert most of their benefical effects by other mechanisms, because smooth muscle cells are the only cell type involved in atherosclerosis which express this type of Ca2’ channels. It is shown that Ca2+ channel blockers modulate signal transduction by interaction with regulatory membrane proteins including purinergic receptors, nucleoside transporter, peripheral benzodiazepine receptors, MDR protein and may be also via receptor operated and/or G protein mediated Ca2+ channels. The relationship between these molecular entities and their corresponding anti-atherogenic cellular effects is poorly understood. However, it has been shown that Ca2’ channel blockers decrease chemotactic migration of monocyte/ macrophages and smooth muscle cells. They also significantly inhibit smooth muscle cell proliferation and extracellular matrix synthesis and may thereby diminish cell adhesion of blood cells and chemical modification of matrix bound LDL. Thus the generation of modified LDL is decreased and this leads to a reduced formation of foam cells. Verapamil and diltiazem based Ca2+ channel blockers can also influence cellular lipid metabolism in monocyte/macrophages and smooth muscle cells due to their lysosomotropic chemical structure. Their molecular structure enables Ca2+ channel blockers to interact with plasma membranes and thereby enhance membrane turnover and fluidity. They have also been shown to interfere with radical metabolism which diminishes radical mediated cell damage. The resulting membrane protective effects possibly delay the progression of the lesions and may improve the integrity of the cytoskeleton as well as stabilizing adjacent membranes. All the present in vitro data of antiatherogenic effects related to Ca*+ channel blockers are in accordance with in vivo studies [X52,153] in various animal models and recently in humans [154]. In conclusion, the results clearly indicate that Ca2 + channel blockers exert anti-atherosclerotic effects at the stage of an early lesion. However, most of these effects are obtained by drug administration in a micromolar range and new drugs have to be developed with higher efficiency at nanomolar concentrations.

Acknowledgments

We gratefully acknowledge Philip D. Henry, Houston, Olga and Yechezkiel Stein, Jerusalem, and David E. Bowyer, Cambridge, for critical reading of the manuscript and their helpful comments. Ross, R., The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314 (1986) 488. Ross, R.. Mechanism of atherosclerosis - a review, Adv.

7

8

9

10

11

Nephrol., 19 (1990) 79. Nilsson, J., Growth factors and the pathogenesis of atherosclerosis, Atherosclerosis, 62 (1986) 185. Gerrity, R.G., The role of the monocyte in atherogenesis, Am. J. Pathol.. 103 (1981) 181. Faggiotto. A., Ross, R. and Harker, L., Studies of hypercholesterolemia in the nonhuman primate, Part 1 and Part 2. Arteriosclerosis, 4 (1984) 323 and 341. Thyberg. J., Hedin, U., Sjolund, M., Palmberg, L. and Bottger, B.A., Regulation of differentiated properties of arterial smooth muscle cells, Arteriosclerosis, 10 (1990) 966. Moncada, S. and Radomski, M.W., The problems and promise of prostaglandin influence in atherogenesis, Ann. N.Y. Acad. Sci., 454 (1985) 121. Vane, J.R., Gryglewski, R.J. and Botting, R.M., The endothelial cell as a metabolic and endocrine organ, Trends Pharmacol. Sci., 8 (1987) 491. Myers, P.R., Minor, R.L., Jr., Guerra. R., Jr., Bates, J.N. and Harrison, D.G., Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrocysteine than nitric oxide, Nature, 345 (1990) 161. Forstermann, U., Miilsch, A.. Biihme, E and Busse, R.. Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived vascular relaxing factor, Circ. Res., 58 (1986) 531. Carter, T.D.. Hallam, T.J.. Cusack, N.J. and Pearson, J.D., Regulation of P*,-purinoreceptor mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br. J. Pharmacol.. 95 (1988) llK1.

12 Boeynaems, J.M. and Pearson, J.D., P, purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms, Trends Pharmacol. Sci., 1 I (1990) 64. 13 Yanagisawa, M., Kurihara, H., Kimura, S., Goto, K. and Masaki. T., Endothelium-derived novel vasoconstrictor peptide endothelin: a possible endogenous agonist for voltage-dependent Ca*’ -channels. In: M. Morad, W. Nayler, S. Kazda and M. Schramm (Eds.), The calcium channel: structure, function and implication, SpringerVerlag, Berlin, Heidelberg, 1988, pp. 575-583. 14 Komura, I., Kurihara, H., Sugiyama, T., Yoshizuam, M.,Takaku, F. and Yazaki, Y., Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells, FEBS Lett., 238 (1988) 249.

128 15 Kugiyama, K., Kerns, S.A., Morrisett, J.D., Roberts, R. and Henry, P.D., Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins, Nature, 344 (1990) 160. 16 Tipping, P.G., Malliavos, J. and Holdswork, S.R., Procoagulant activity expression by macrophages from atheromatous vascular plaques, Atherosclerosis, 79 (1989) 237. 17 Bevilacqua, M.P., Schleef, R.R., Gimbrone, M.A., Jr. and Loskutoff, D.J., Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin-1, J. Clin. Invest., 160 (1986) 587. 18 Pober, J.S., Cytokine-mediated activation of vascular endothelium, Am. J. Pathol., 133 (1988) 426. 19 Matsubara, T., Ziff, M., Increased superoxide anion release from human endothelial cells in ‘response to cytokines, J. Immunol., 137 (1986) 3295. 20 Winther, K., Warberg, J. and Knigge, U., The effect of intrinsic sympathomimetic activity on platelet aggregation and fibrinolytic acitvity, Curr. Opin. Cardiol., 3 (Suppl. 2) (1988) 129. 21 Grulich-Henn, J. and Miiller-Berghaus, G., The role of vascular endothelial cells in the regulation of fibrinolysis, Z. Kardiol., 78 (1989) 25. 22 Rowland, F.N., Donovan, M.J. and Picciano, P.T., Fibrin mediated vascular injury. Identification of fibrin peptides that mediate endothelial cell reaction, Am. J. Pathol., 117 (1984) 418. 23 Hajjar, K.A., Gavish, D., Breslow, J.L. and Nachman, R.L., Lipoprotein (a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis, Nature, 339 (1989) 303. 24 Johnsson, H., Effects by nifedipine on platelet function in vitro and in vivo, Thromb. Res., 21 (1981) 523. 25 Kiyomoto, A., Sasaki, J., Odawara, A. and Morta, T., Inhibition of platelet aggregation by diltiazem, Circ. Res., 52 (1983) 115. 26 Metha, J., Metha, P. and Ostrowski, N., Calcium blocker diltiazem inhibits platelet activation and stimulates vascular prostacyclin synthesis, Am. J. Med. Sci., 291 (1986) 20. 27 Metha, J., Metha, P., Ostrowski, N. and Crews, F., Effects of verapamil on platelet aggregation, ATP release and thromboxane generation, Thromb. Res., 30 (1983) 469. 28 Baldenkov, G.N., Akopov, SE., Ryong, L.H. and Orekhov, A.N., Prostacyclin, thromboxane A, and calcium antagonists: effects on atherosclerotic characteristics of vascular cells, Biomed. Biochim. Acta, 47(10-11) (1988) 324. 29 Fritschka, E., Kribben, A., Distler, A. and Philipp, T., Inhibiton of aggregation and calcium influx of human platelets by nifedipine, J. Cardiovasc. Pharmacol., 9 (1987) 985. 30 Schror, K., Plattchenhyperreaktivitat und Arachidonsaurestoffwechsel in der Atherosklerose. Calciumantagonisten und Atherosklerose, Medikon Verlag, Miinchen, 1989, p. 21. 31 Ferri, C., de Angelis, C., del Porto, M.A., LeonettiLuparini, R., Giarrizzo, C., Santucci, A. and Balsano, F.,

32

33

34

35 36

37

38

39 40

41

42

43

44

45 46

47

Effect of nicardipine on angiotensin II platelet content, J. Hypertens., Suppl. 6 (1988) 117. Block, L.H., Emmons, L.R., Vogt, E., Sachinidis, A., Vetter W and Hoppe, J., Ca2+-channel blockers inhibit the action of recombinant platelet-derived growth factor in vascular smooth muscle cells, Proc. Nat]. Acad. Sci. USA, 86 (1989) 2388. Tedgui, A., Chiron, B. and Curmi, P., Effect of nicardipine and verapamil on in vitro albumin transport in rabbit thoracic aorta, Arteriosclerosis, 7 (1987) 80. Strohschneider, T. and Betz, E., Densitometric measurement of increased endothelial permeability in arterosclerotic plaques and inhibition of permeability under the influence of twp calcium antagonists, Atherosclerosis, 75 (1989) 135. VanHoutte, P.M., Cazf-antagonists and vascular disease, Ann. N.Y. Acad. Sci., 522 (1988) 380. Grodzinska, L., Basista, M., Slawinski, M., Swies, J., Stachura, J. and Ohlrogge, R., Nifedipine stimulated release of prostacyclin-like substance in normal and atherosclerotic animals, Arzneim. Forsch., 37 (1987) 412. Gleerup, G. and Winther, K., Differential effects of non-specific beta-blockade and calcium antagonism on blood-clotting mechanism, Am. J. Med., 86 (1989) 127. Senegelov, H. and Winther, K., Effect of felodipine, a new calcium channel antagonist, on platelet function and fibrinolytic activity at rest and after exercise, Eur. J. Clin. Pharmacol., 37 (1989) 453. Winther, K., Scandiavian Adalate Conference, Copenhagen, 1990. Fleckenstein, A., Frey, M., Zorn, J. and FleckensteinGrim, G., The role of calcium in the pathogenesis of experimental arteriosclerosis, Trends Pharmacol. Sci., 8 (1987) 496. Akopov, S.E., Orekhov, A.N., Tertov, V.V., Khashimov, K.A., Gabrielyan, E.S. and Smirnov, V.N., Stable analogues of prostacyclin and thromboxane A, display contradictory influences on atherosclerotic properties of cells cultured from human aorta: The effect of calcium antagonists, Atherosclerosis, 72 (1988) 245. Stasch, J.P., Kazda., Endothelin-1 induced vascular contractions: interactions with drugs affecting the calcium channel, J. Cardiol. Pharmacol., 13 (1989) 561. Le Breton, G.C., Dinerstein, R.J., Roth, L.J. and Feinberg, F., Direct evidence for intracellular divalent cation redistribution associated with platelet shape change, Biochem. Biophys. Res. Commun., 71 (1976) 362. Feinstein, Feikers, J. and Fraiser, C., An analysis of the mechanism of local anesthetic inhibition of platelet aggregation and secretion, J. Pharmacol. Exp. Ther., 197 (1976) 215. Ardlie, N.G., Calcium ions, drug action and platelet function, Pharmacol. Ther., 18 (1982) 249. Parthasarathy, S., Wieland, E. and Steinberg, D., A role of endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein, Proc. Nat]. Acad. Sci. USA, 86 (1989) 1046. Parthasarathy, S., Steinbrecher, U.P., Barnett, J., Witzum,

129

48

49

50

51 52

53

54

J.L. and Steinberg D., Essential role of phospholipase A, activity in endothelial-induced modification of low density lipoprotein, Proc. Natl. Acad. Sci. USA, 82 (1985) 3000. Quinn, M.T., Parthasarathy, S. and Steinberg, D.. Lysophosphatidylcholine: A chemotactic factor for human monocytes and its potential role in atherosclerosis, Proc. Natl. Acad. Sci. USA, 85 (lY88) 2805. Palinski, W., Rosenfeld, M.E., Yla-Herttuala, S.. Gurtner, G.C.. Socher, S.S.. ButleT, S.W., Parthasarathy, S.. Carew, T.E.. Steinberg, D. and Witzum, J.L., Low density lipoprotein undergoes oxidative modification in vivo. Proc. Natl. Acad. Sci. USA, 86 (1989) 1372. Steinberg, D.. Parthasarthy, S.. Carew, T.E., Khoo, J.C. and Witzum. J.L.. Beyond cholesterol.. Modification of low-density lipoprotein that increases its atherogenecity. N. Engl. J. Med., 320 (1989) 915. Brown, M.S. and Goldstein, J.L.. Scavenging for receptors, Nature. 343 (1990) 508. Sparrow. C.P.. Parthasarathy, S. and Steinberg. D.A.. Macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. J. Biol. Chem., 264 (1989) 2599. Hinsbergh. V.W.M., Scheffer, M.A.. Havehest, L. and Kempen. J.M.. Role of endothelial cells and their products in the modification of low density lipoprotein, Biochim. Biophys. Acta, 49 (1986) 878. Brown, M.S. and Goldstein, J.L., Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Annu. Rev. Biochem.. 52 (1983)

62 Trail], 63

64

65

66

67 6X

69

iL5.

55 Brown. MS., Ho, Y.K. and Goldstein. J.L., The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters, J. Biol. Chem.. 255 (1980) 9344. 56 Havel, R.J.. Role of triglyceride-rich lipoproteins in progression of atherosclerosis, Circulation. 81 (1990) 694. 57 Lindquist, P.. Osthend-Lindquist. A.M.. Witzum, J.L. and Steinberg. D., Role of lipoprotein lipase in the metabolism of triglyeride-rich lipoproteins by macrophages, J. Biol. Chem., 258 (1983) 9086. 5X Fogelman. A.M., Seager, J., Groopman. J.A., Haberland, M.E.. Edwards, P.A. and Golde. D.W., Lymphokines secreted by an established lymphocyte line modulate receptor-mediated endocytosis in macrophages derived from human monocytes, J. Immunol.. 131 (1983) 2368. 59 Van Lenten. B.J.. Fogeiman, A.M., Seager. J.. Ribi. E., Haberland. M.E. and Edwards, P.A., Bacterial endotoxin selectively prevents the expression of scavenger-receptor activity on human monocyte-macrophages, J. Immunol., 134 (1985) 371x. 60 Kraemer. F.B.. Tavangar, K., Gandjei, R.K., Kilrew and Behr, S.R.. Effects of activation on lipid and lipoprotein metabolism in murine macrophages. Arteriosclerosis. 10 (1990) 8. 61 tiims Ziegler-Heitbrock. H.W., The biology of the monocyte system. Eur. J. Cell Biol., 49 (1989) 1.

70

71

72

73

74

75

K.N. and Wick, G., Lipids

and lymphocyte

func-

tion, Immunol. Today, 5 (1984) 70. Nakao, J., Ooyama, T., Chang, W.C., Churota, S. and Orimo, H., Platelets stimulate aortic smooth muscle cell of 12.I.-hydroxymigration in vitro - involvement 5.8,10,14-eicosatetranoic acid. Atherosclerosis. 43 (1982) 143. Nomoto, A., Mutoh, S., Hagihara, H. and Yamaguchi. I., Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nivaldipine, Atherosclerosis, 72 (1988) 213. Pennington, J.E., Kemmerich, B., Kazanjian, P.H., March, J.D. and Boerth, L.W., Verapamil impairs human neutrophil chemotaxis by a non calcium mediated mechanism,. J. Lab. Clin. Med.. 108 (1986) 44. Williamson, K.C., Tauber, A.I. and Navarro, J.. Nisoldipine inhibits formyl-methinyl-leucyl-phenylalanine receptor-coupled calcium transport in human neutrophils, J. Leukocyte Biol., 42 (1987) 239. Springer. T.. Adhesion receptors of the immune system. Nature, 346 (1990) 425. Schmitz. G., Robenek, H.. Beuck. M.. Krause, R.. Schurek. A. and Niemann, R.. Ca’+-antagonsits and ACAT inhibitors promote cholesterol efflux from macrophages by different mechansims. 1. Characterization of cellular lipid metabolism, Arteriosclerosis, 8 (1988) 46. Stein. O., Leitersdorf. E. and Stein, Y.. Verapamil enhances receptor-mediated endocytosis of low density lipoproteins by aortic cells in culture. Arteriosclerosis, 5 (1985) 35. Etingin, O.R. and Hajjar, D.J.. Nifedipine increases cholesteryl ester hydrolytic activity in lipid-laden arterial smooth muscle cells, J. Clin. Invest.. 75 (1985) 1554, Orekhov. A.N., Tertov. V.V., Khashimov, K.A., Kudryashov. S.A. and Smirnov. V.N., Antiatherosclerotic effects of verapamil in primary culture of human aortic intimal cells, J. Hypertens.. 4 (1986) 153. Orekhov. A.N.. Baldenkov. G.N.. Tertov, V.V., Ryong. L.H.. Kozlov. S.G.. Lyakishev. A.A.. Tkachuk, V.A., Ruda. M.Y. and Smirnov, V.N.. Cardiovascular drugs and atherosclerosis: effects of calcium antagonists, betablockers and nitrates on atherosclerotic characteristics of human aortic cells. J. Cardiol. Pharmacol.. 12 (1988) 566. Etingin, O.R. and Hajjar, D.J., Calcium channel blockers enhance cholesteryl ester hydrolysis and decrease totill cholesterol accumulation in human aortic tissue, Circ. Res., 66 (1990) 185. Yatsu, F.M.. ,4lam. R. and Alam, S.S.. Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil, B&him. Biophys. Acta. X47 (1985) 77. Daugherty, A., Rateri, D.L.. Schonfeld, Cr. and Sobel, B.E., Inhibition of cholesteryl ester deposition in macrophages by calcium entry blockers: an effect dissociable from calcium entry blockade. Br. J. Pharmacol.. 33 (1987) 2377.

130 76 Stein, O., Leitersdorf, E. and Stein, Y., Verapamil enhances receptor-mediated endocytosis of low density lipoproteins by aortic cells in culture, Arteriosclerosis, 5 (1985) 35. 77 Corsini, A., Granata, A., Fumagalli, R. and Paoletti, R., Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human heptoma cell line Hep G2, Pharmacol. Res. Commun., 18 (1986) 1. 78 Ranganathan, S., Harmony, J.A.K. and Jackson, R.L., Effects of CaZC blocking agents on the metabolism of low density lipoproteins in human skin fibroblasts, Biochem. Biophys. Res. Commun., 107 (1982) 217. 79 Mori, S., Ito, H. and Yamamoto, K., Effects of calcium antagonists on low density lipoprotein metabolism in human arterial smooth muscle cells, Tohoku J. Exp. Med., 154 (1988) 329. 80 Stein, 0. and Stein, Y., Effect of verapamil on cholestetyl ester hydrolysis and reesterification in macrophages, Arteriosclerosis, 7 (1987) 578. 81 Paoletti, R., Bernini, F., Fumagalli, R., Allorio, and Corsini, A., Calcium antagonists and low density lipoprotein receptors, Ann. N.Y. Acad. Sci., 522 (1988) 390. 82 Campbell, J.H. and Campbell, G.R., Endothelial cell influences on vascular smooth muscle phenotype, Annu. Rev. Physiol., 48 (1986) 295. 83 Hedin, U., Bottger, B.A., Forsberg, E., Johansson, S. and Thyberg, J., Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells, J. Cell Biol., 107 (1988) 307. 84 Benzonana, G., Skalli, 0. and Gabbiani, G., Correlation between the distribution of smooth muscle or non muscle myosins and o-smooth muscle actin in normal and pathological soft tissues, Cell Motil. Cytoskeleton, 11 (1988) 260. 85 Campbell, J.H., Reardon, M.F., Campbell, G.R. and Nestel P.J., Metabolism of atherogenic lipoproteins by smooth muscle-cells of different phenotype in culture, Arteriosclerosis, 5 (1985) 318. 86 Deuel, T.F., Senior, R.M., Huang, J.S. and Griffin, G.L., Chemotaxis of monocytes and neutrophils to platelet-derived growth factor, J. Clin. Invest., 69 (1982) 1049. 87 Heldin, C.-H. and Westermark, B., Platelet-derived growth factor: three isoforms and two receptor types, Trends Genet., 5 (1989) 108. 88 Habenicht, A.J.R., Glomset, J.A., King, W.C., Nist, C., Mitchell, CD. and Ross, R., Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet-derived growth factor, J. Biol. Chem., 256 (1981) 12329. 89 Goldstein, J.L. and Brown, M.S., Regulation of the mevalonate pathway, Nature, 343 (1990) 425. 90 Sejersen, T., Betsholtz, C., Sjolund, M., Heldin, C-H., Westermark, B. and Thyberg, J., Rat skeletal myoblasts and arterila smooth muscle cells express the gene for the A chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein, Proc. Natl. Acad. Sci. USA, 83 (1986) 6844.

91 Suzuki, H., Shibano, K., Okane, M., Kono, I., Matsui, Y., Yamane, K. and Kashiwagi, H., Interferon-gamma modulates messenger RNA levels of c-sis (PDGF-B chain), PDGF-A chain and IL-lp genes in human vascular endothelial cells, Am. J. Pathol., 134 (1989) 35. 92 Loppnow, H. and Libby, P., Proliferating or interleukinl activated human vascular smooth muscle cells secrete copious interleukin 6, J. Clin. Invest., 85 (19904 731. 93 Berk, B.C. and Alexander, R.W., Vasoactive effects of growth factors, Biochem. Pharmacol., 38 (1989) 219. 94 Palmberg, L., Claesson, H.-E. and Thyberg, J., Effects of leukotrienes on phenotypic properties and growth of arterial smooth muscle cells in primary culture, J. Cell Sci., 93 (1989) 403. 95 Pomerantz, K.B. and Hajjar, D.P., Eicosanoids in regulation of arterial smooth muscle cell phenotype, proliferative capacity, and cholesterol metabolism, Arteriosclerosis, 9 (1989) 413. 96 Sinzinger, H., Is human arterial smooth muscle cell proliferation regulated by prostaglandins? Exp. Pathol., 17 (1979) 354. 97 Jonzon, B., Nilsson, J. and Fredholm, B.B., Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells, J. Cell Physiol., 124 (1985) 451. 98 Nilsson, J. and Olsson, A.G., Prostaglandin E, inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet-derived growth factor, Atherosclerosis, 53 (1984) 77. 99 Huang, J.S., Huang, S.S. and Denel, T.F., Specific covalent binding of platelet-derived growth factor to human plasma a2-macroglobulin, Proc. Natl. Acad. Sci. USA, 81 (1984) 342. 100 Nilsson, J., Sjolund, M., Palmberg, L., Von Euler, A.M., Jonzon, B. and Thyberg, J., The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation, Atherosclerosis, 58 (1985) 109. 101 Betz, E., Hammerle, H. and Strohschneider, T., Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas, Res. Exp. Med., 185 (1985) 325. 102 Van Valen, R.G., Deacon, R.W., Farley, C., Melden, M.K., Uhl H.F., Saunders, R.N. and Handley, D.A., Antiproliferative effect of calcium channel blockers PN 200-110 and PY 108-068 in the rat carotid model of ballon catheterization, Fed. Proc., 44 (1985) 737. 103 Saito, Y., Fujiyama, Y., Shirai, K. and Yoshida, S., Effects of nifedipine on lipid metabolism in smooth muscle cells, In: Lichtlen (Ed.), Proc. 6th Int. Adalat Symp., Excerpta Medica, Amsterdam, 1983, pp. 479-483. 104 Nomoto, A., Hirosumi, J., Sekuguchi, C., Mutoh, S., Yamaguchi I. and Aoki, H., Antiatherogenic activity of FR 34235 (nivaldipine), a potent calcium antagonist, Atherosclerosis, 64 (1987) 255. 105 Stein, O., Halperin, G. and Stein, Y., Long-term effects of verapamil on aortic smooth muscle cells cultured in the presence of hypercholesterolemic serum, Arteriosclerosis, 7 (1987) 585.

131 106 Heider, J.G., Weinstein, D.B., Ickens, C.E., Lan, S. and Su, CM., Anti-atherogenic activity of the calcium channel blocker isradipine (PN 200-110): a novel effect on matrix synthesis independent of calcium channel blockade, Transpl. Proc., 29 (1987) 96. 107 Betz, E., The effect of calcium antagonists on intimal cell proliferation in atherogenesis. Ann. N.Y. Acad. Sci., 522 (1988) 399. 108 Jackson, C.L., Bush, R.L. and Bowyer, D.E., Inhibitory effect of calcium channel antagonists on ballon-induced arterial smooth muscle cell proliferation and lesion size. Atherosclerosis, 69 (1988) 115. 109 Absher. M.P., Baldor, L. and Warshaw, D.M., Effects of nitrendipine on growth activity in cultured vascular smooth muscle cells, J. Cardiovasc. Pharmacol., 12 Suppl. 4 (1988) 104. 110 Jackson, CL.. Bush, R.C. and Bowyer, D.E., Mechanism of antiatherogenic action of calcium antagonists, Atherosclerosis, 80 (1989) 17. 111 Cheung, W.T., Shi, M.M., Young, J.D. and Lee, CM.. Inhibition of radioligand binding to A, adenosine receptors by Bay K 8644 and nifedipine. Biochem. Pharmacol., / 36 (1987) 2183. 112 Fiilop, T.. Jacob, M.P. and Varga, Z.S.. Effect of elastin peptides on human monocytes: Cal+ mobilization. stimulation of respiratory burst and enzyme secretion, Biochem. Biophys. Res. Commun., 141 (1986) 92. 113 Orekhov, A.N., Andreeva, E.R., Shekhonin, B.V., Tertov, V.V. and Smirnov, V.N., Content and localization of fibronectin in normal intima, atherosclerotic plaque, and underlying media of human aorta, Arteriosclerosis, 53 (1984) 213. 114 Wight, T.N.. Cell biology of arterial proteoglycans. Arteriosclerosis, 9 (1989) 1. 115 Camejo. G.. The interaction of lipids and lipoproteins with the extracellular matrix of arterial tissue: its possible role in atherogenesis, Adv. Lipid Res.. 19 (1982) 1. 116 Dietrich, J.W. and Duffield, R., Effects of the calcium antagonist verapamil on in vitro synthesis of skeletal collagen and non-collagen proteins, Endocrinology. 105 (lY7Y) 116X. 117 Weinstein, D.B. and Heider, J., Antiatherogenic properties of calcium antagonists. Am. J. Cardiol., 59 (1987) 163B. 118 Shi, B. and Tien. H.T.. Action of calcium channel and beta-adrenergic blocking agents in bilayer lipid membranes, Biochim. Biophys. Acta, 859 (1986) 123. I19 Shiridi, F. and Robal, J.. The influence of calcium channel blockers on superoxide anions, Pharmacol. Res. Commun., 20 (1988) 13. 120 Ondrias. K., Misik, V.. Gerbel, D. and Stasko. A., Lipid peroxidation of phosphatidylcholine liposomes depressed by calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine, Biochim. Biophys. Acta. 1003 (1989) 238. 121 Herbette. L.G.. Exve, Y. and Rhodes, D.G.. Interaction of 1.4.dihydropyridine calcium channel antagonists with

122

123

124

125

126

127

128 129

130

131

132

biological membranes: lipid bilayer partitioning could occur before drug binding to receptor, J. Mol. Cell Cardiol., 21 (1989) 187. Weiss, K., Calcium antagonists: anti-atherosclerotic effect by modification of the prostaglandin system. Wien. Klin. Wochenschr., 4:lOO (1988) 718. Janero, D.R., Burghardt, B. and Lopez, R., Protection of cardiac membrane phospholipid against oxidative injury by calcium antagonists, Biochem. Pharmacol., 37 (1988) 4197. Mak, 1.T. and Weglicki, W.B., Comparative antioxidant activities of propranolol, nifedipine. verapamil and diltiazem against sarcolemmal membrane lipid peroxidation, Circ. Res., 66 (1990) 1449. Bean, B.P. and Rios, E., Nonlinear charge movement in mammalian cardiac ventricular cells: components from Na and Ca channel gating, J. Gen. Physiol.. 94 (1989) 65. Schmitz, G., Beuck, M., Fischer, H., Nowicka. G. and Robenek H., Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoproteinmediated cholesterol efflux in macrophages. J. Lipid Res.. 31 (1990) 1731. Slotte, P., Hedstrom, G., Rannstrom. S. and Ekman S.. Effects of sphingomyelin degradation on cell cholesterol oxidizability and steady-state distribution between the cell surface and the cell interior, Biochim. Biophys. Acta, 985 (1989) 90. Phair, R.D., Cellular calcium and atherosclerosis: a brief review. Cell Calcium, 9 (1988) 275. Takahasi, M., Seagar, M.J.. Jones, J.F., Reber, B.F.X. and Catterall, W.A., Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle, Proc. Natl. Acad. Sci. USA. 84 (1987) 5478. Schmitz, G.. Hankowitz, J.. Brennhausen. B. and Schmutte. C.. Der Eintlug von Calcium-Antagonisten auf den zellularen Lipidstoffwechsel, Arzneim.-Forsch./Drug Res.. JO (1990) 366. Harootunian. A.T.. Kao, J.P.Y., Paranjape. S. and Tsien. R.Y., Generation of calcium oscillations in fihroblasts by positive feedback between calcium and IPI. Science. 251 (1991) 75. Birnbaumer. L., Abramowitz, J. and Brown. A.M.. Receptor-effector coupling by G proteins. Biochim. Biophys. Acta, 1031 (1990) 163.

133 Rasmusson, H.. The complexities of intracellular calcium signalling. Hoppe-Seyler’s Z. Biol. Chem.. 371 (1990) 191. 134 Striessnig. J., Gall, A., Moosburger, K. and Glossmann. H.. Purified calcium channels have three allosterically coupled drug receptors, FEBS Lett., 197 (1986) 204. 135 Cantor. E.H., Kenessy, A., Semenuk. G. and Spector, S.. Interaction of calcium channel blockers with non-neuronal benzodiazepine binding sites. Proc. Natl. Acad. Sci. USA, XI (1984) 1549. 136 Gottesman, M. and Pastan, I., The multidrug transporter, a double-edged sword, J. Biol. Chem.. 263 (1988) 12163. 137 Burnstock. G.. A basis for distinguishing two types of purinergic receptor. In: Straub. R.W. and Bolis. L. (Eds.).

132

138

139

140

141

142

143

144

145

Cell Membrane Receptors for Drugs and Hormones: a mulitdisciplinary approach Raven Press, New York, 1978, pp. 107-118. Haslam, R.J and Rosson, G.M., Effects of adenosine on levels of cyclic adenosine-3’,5’-monophosphate in human blood platelets in relation to adenosine incorporation and platelet aggregation, Mol. Pharmacol., 11 (1975) 528. Edvinsson, L. and Fredholm, B.B., Characterization of adenosine receptors in isolated cerebral arteries of cat, Br. J. Pharmacol., 80 (1983) 631. Collis, M.J., In: T.W. Stone (Ed.), Purines: Pharmacology and physiological role, Macmillan, London, 1985, pp. 75-84. Kennedy, C., In: G. Burnstock and S.G. Griffith (Eds.), Nonadrenergic Innervation of Blood Vessels. Vol. 1, CRC Press, Boca Raton, FL, 1988, pp. 65-76. Cronstein, B.N., Kramer, S.B., Rosenstein, E.D., Korchak H.M., Weissmaynn, G. and Hirschhorn, R., Occupancy of adenosine receptors raises cyclic AMP alone and in synergy with occupancy of chemoattractant receptors and inhibits membrane depolarization, Biochem. J., 252 (1988) 709. Eppel, B.A, Newell, A.M. and Brown, E.J., Adenosine receptors are expressed during differentiation of monocyte to macrophages in vitro: Implications for regulation of phagocytosis, J. Immunol., 143 (1989) 4141. Schultz, L.A., Kammer, G.M., Rudolph, S.A., Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells, FASEB J., 2 (19881 244. Hafely, W., Kyburz, E., Gerecke, M. and Miihler, H., Recent advances in the molecular pharmacology of benzodiazepine receptors and in the structure-activity relationships of their agonists and antagonists, Adv. Drug Res., 14 (19851 165.

146 Verma, A. and Snyder, S.H., Peripheral type benzodiazepine receptors, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 307. 147 Besman, M.J., Yanagibashi, K., Lee, T.D., Kawamura, M., Hall P.F. and Shively, J.E., Identification of des-(GlyBe)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: Stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor, Proc. Natl. Acad. Sci. USA, 86 (1989) 4897. 148 Mantione, CR., Goldman, M.E., Martin, B., Bolger, G.T. and Luddens, H.W., Purification and characterization of an endogenous protein modulator of radioligand binding to “peripheral type” benzodiazepine receptors and dihydropyridine Ca2’ -channel binding sites, Biochem. Pharmacol., 37 (1988) 339. 149 Beaumont, K., Skowronski, R., Vangh, D.A. and Fanestil, D.D., Interaction of lipids with peripheral type benzodiazepine receptors, Biochem. Pharmacol., 37 (1988) 1009. 150 Rampe, D. and Triggle, D.J., Benzodiazepines and calcium channel function, Trends Pharmacol. Sci., 11 (1986) 461. 151 Zernig, G., Widening potential for Ca2+ antagonists: non-L-type Ca 2+ channel interaction, Trends Pharmacol. Sci., 11 (1990) 38. 152 Henry, P.D and Bently, K.I., Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine, J. Clin. Invest., 68 (1981) 1366. 153 Henry, P.D., Calcium anatgonists as anti-atherosclerotic agents, Arteriosclerosis, 10 (19901 963. 154 Lichtlen, P.R., Hugenholtz, P.G., Rafflenbeul, W., Hecker H., Jest, S. and Deckers, J.W., Retardation of angiographic progression of coronary artery disease by nifedipine. Results of International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT), Lance& 335 (1990) 1109.