Pat hophysi ol ogi c Sti mul i in At heroscl erosi s
Mi chael A. Gi mbr one, Jr., MD V a s c u l a r e n d o t h e h m , t h e ~ l i ni ng o f t h e c a r d i o v a s c u l a r s y s t e m, i s a n I mp o r t a n t f u n c t i o n a l c o mp o n e n t o f t h e Mo o d v e s s e l wag, a c t i v e l y ~ i n n o r ma l v a s c u l a r physi ol - ogy a s we l l a s t h e p a t h o g e e e s i s o f v a s c u l a r di s- e a s e s s uc h a s / h e r o s c l e r o e i L The l ocal i z ed mo d u l a t i o n o f v a s c u l a r ~ m t o a non. a d a p t i v e f u n c t i o n a l s t a t e c a n b e t e r me d " e ndo- t h e l i a l dy s f unc t i on. " Thi s a r t i c l e p r o v i d e s a b r i e f overview o f endot hehl dysfunction, especi al l y a s i t r e l a t e s t o mo n o n u c l e a r l e u k o c y t e r e c r u i t . me n t dur i ng a t h e c o s c l e ~ U c l esi on f o r ma t i o n . Potenti al di agnosti c a n d t herapeut i c i mpl i cati ons are al so consi der ~. ( Am J Ca r d k 4 1 9 9 5 ; 7 5 : 6 7 B - 7 0 B ) From t he Vascul ar Research Division, Bri gham and Women' s Hospital, Boston, Massachusetts. The original studies described in this article were conduct ed in t he Vascul ar Research Division of the Depar t ment of Pathology at t he Bri gham and Women' s Hospital and were support ed primarily by research grants from t he National Heart , Lung, and Blood Inst i t ut e and t he Massachusetts Affiliate of tlae Ameri can Hear t Association. Address for reprints: Michael A. Gi mbrone, Jr., MD, Vascul ar Research Division, Bri gham and Women' s Hospital, 221 Long- wood Avenue (LMRC-4), Boston, Massachuset t s 02115-5817. N onadaptive interactions of cellular and macromolecular components of circulat- ing blood with the arterial wall play an important role in the pathogenesis of atherosclero- sis and coronary thrombosis. Increasing evidence indicates that alteration in the functional proper- ties of the vascular endothelial lining, or endothe- lial dysfunction, may underlie certain of these nonadaptive interactions and thus contribute to the initiation, progression, and clinical complica- tions of atherosclerotic vascular disease. Although the involvement of endothelium in the atherosclerotic process has been recognized since the time of Virchow, 1 many relevant aspects of its biology and pathobiology have been appreciated only recently, z We now know that this single-cell- thick tissue is, in fact, multifunctional and can directly influence circulating blood components as well as other cell types within the blood vessel wall. 3,4 In addition, this vital interface can undergo dramatic functional alterations in response to vari- ous pathophysiologic stimuli through the process of endothelial activation. 5~ This article highlights some recent insights into the stimuli and consequences of endothelial dys- function that relate to the pathogenesis of athero- sclerosis. In particular, it focuses on the cellular and molecular mechanisms by whi ch activated arterial endothelium selectively recruits mono- nuclear leukocytes. This recruitment process, analo- gous to recently described endothelium-dependent mechanisms operative in acute and chronic inflam- mation, 9,t appears to be a key step in the pathogen- esis of atherosclerotic lesions. How these endothe- lial mechanisms relate to the complex interplay of biochemical risk factors in lesion initiation and progression, and how they are potentially utilized as targets for diagnostic strategies and therapeutic interventions, are also considered. THE AT HE RO- E L AM HY P OT HE S I S E n d o t h e i l ~ m e c h a n i s m s o f l e u - k o c y t e r e c r b ; i , , , e n t I n at her ogem~l s: Perhaps the earliest morphologically detectable cellular A SYMPOSIUM: ATHEROTHROMBOSIS 67B event in at herogenesi s is t he adherence of circulat- ing blood monocyt es to t he intact intimal surface of large arteries, u-13 These adher ent cells t hen mi- grate across t he endot hel i um into t he subendo- thelial intima, where they t end to accumulate, undergo limited replication, and t ransform into lipid-laden foam cells. Once present in t he intima, this differentiating monocyt e- macr ophage popula- tion accumulates cholesterol esters (the lipid "hall- mark" of t he early fatty streak lesion) and pro- mot es lesion progression t hrough local generat i on of cytokines (e.g., interleukin-1, t umor necrosis factor); growth factors (e.g., platelet-derived growth factor, fibroblast growth factor, hepari n-bi ndi ng epidermal-growth-factor [EGF]-like growth fac- tor), and various procoagul ant and fibrinolytic component s, eicosanoids, and toxic oxygen prod- ucts. 2 Recently, considerable at t ent i on has been focused on t he presence, in more-devel oped le- sions, of T lymphocytes t hat have t he capacity to generat e ot her cytokines (e.g., i nt erferon-gamma, interleukin-4) t hrough bot h i mmune- and nonim- mune-t ri ggered pathways. These lymphocyte-de- rived product s add furt her complexity to t he local cytokine milieu and t he pot ent i al activation states of vessel wall cells (endot hel i um and smoot h muscle) and ot her component s of t he developing atherosclerotic lesion. 14 The localized, leukocyte-selective nat ure of this mononucl ear recrui t ment process suggests that e ndot he l i um- de pe nde nt adhesi on mechani sms analogous to t hose recently described in acute and chronic inflammation might be responsible. 15 This inference led to t he hypothesis t hat these localized mononucl ear l eukocyt e- endot hel i um interactions reflect specific mol ecul ar changes in t he adhesive propert i es of t he endot hel i al surface, involving inducible endot hel i um-l eukocyt e adhesi on mol- ecules (ELAM) expressed in atherosclerotic le- sions ( ATHERO- ELAM) . 16 According to this hy- pothesis, a candi dat e ATHERO- ELAM should: (1) support mononucl ear (but not necessarily poly- morphonucl ear) leukocyte adhesion; (2) be induc- ibly expressed on t he endot hel i al surface; and (3) be det ect abl e in early atherosclerotic lesions (or in areas with a predisposition for developing lesions). The experi ment al search for molecules satisfy- ing t hese criteria was under t aken in t he rabbit, a species in which dietary and genetic model s of atherosclerosis are well described.16 Initially, leuko- cyt e- endot hel i um interactions were examined in vitro, using cul t ured normal rabbit aortic endot he- lial cells. Tr eat ment of t hese endot hel i al monolay- ers with a nonspecific activator, such as bacterial endotoxin, caused t he surface to become hyperad- hesive, a change that was detectable following incubation with blood monocytes and monocyte- like cell lines. This endot hel i al surface hyperadhe- sion, which was dependent on prot ei n synthesis, was det ect abl e after a lag of 1-2 hours, peaked within 6-12 hours, and t hen remai ned manifest for at least 96 hours. Muri ne monocl onal antibodies to these endotoxin-activated rabbit endothelial cells det ect ed various inducible surface ~ntigens, some of which exhibited a similar t emporal profile to that of t he adhesive surface change for blood leuko- cytes. In adhesi on assays, pr et r eat ment of activated endot hel i al monolayers with saturating concentra- tions of certain of these monoclonal antibodies significantly inhibited mononucl ear leukocyte at- t achment . Comparat i ve studies with various types of leukocytes showed that this inhibition was selec- tive for mononucl ear, but not polymorphonuclear, leukocyte at t achment . These in vitro studies thus identified an inducible adhesion molecule that could selectively support mononucl ear leukocyte adhesi on to activated arterial endothelial cells, thereby satisfying t he first 2 of t he experimental criteria for an ATHERO- ELAM. The expression of this inducible, mononuclear leukocyte-selective adhesi on molecule was then examined during experimental atherogenesis. In rabbits fed a 1% cholesterol diet, and in Watanabe heritable hyperlipidemic rabbits, which have se- vere hypercholesterolemia, specific monoclonal an- tibody staining was localized to aortic endothelium covering foam cell-rich intimal lesions. Staining was also evident at various stages of lesion develop- ment, ranging from focal regions with very small intimal accumul at i ons of foam cells to near- circumferential lesions with abundant foam cells. Staining oft en ext ended beyond t he edges of inti- mal lesions; however, in t he same hypercholesterol- emic animals, endot hel i um in adjacent, uninvolved regions of t he aorta did not stain. These in vivo i mmunohi st ochemi cal observations thus satisfied t he third experimental criterion for an ATHERO- ELAM, its expression by endot hel i um duri ng early lesion format i on and progression. The mol ecul ar characterization of this rabbit ATHERO- ELAM t hrough a combination of immu- nochemical and mol ecul ar cloning approaches re- vealed its homology to a human leukocyte adhesi on molecule, intercellular adhesion molecule-110 (pre- viously identified as an inducible endot hel i al recep- tor for lymphocytes), 17 and expression cloned from a cytokine-induced endot hel i al library as vascular cell adhesi on molecule-1 (VCAM-1). 18 This cyto- 6811 THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 75 FEBRUARY 23, 1995 ki ne-act i vat ed gene is a member of t he i mmuno- globulin superfami l y and is expressed on t he sur- face of human and rabbit vascular endot hel i um in at least 2 mol ecul ar forms, presumabl y deri ved by al t ernat i ve splicing. 19 Both forms can i nt eract with het er odi mer i c integrin recept or, t er med very late antigen-4 (VLA-4) or et4131, a member of t he 131 subfamily of integrins t hat is differentially ex- pressed on cert ai n leukocytes, including bl ood monocyt es and lymphocytes, but not on polymor- phonucl ear leukocytes. 2,21 This mat chi ng of endo- t hel i um-expressed VCAM- 1 in mononucl ear leuko- cyt e- expr es s ed VLA- 4 t her ef or e provi des a funct i onal count er r ecept or pai r capable of medi at - ing a selective adhesi on event. Interestingly, when t he rabbit aort a is carefully exami ned in a di et ary model of at herogenesi s, 22 endot hel i al expression of VCAM-1 is det ect abl e before mononucl ear l eukocyt e r ecr ui t ment into t he art eri al wall. Since one of t he earliest changes during atherogenesis in cholesterol-fed animal mod- els appears to be t he focal accumul at i on and oxidative modification of low density l i poprot ei n in t he intima, 23,24 it is reasonabl e to hypot hesi ze t hat oxidatively modi fi ed low density lipoprotein, or one of its component s, is an initial stimulus for VCAM-1 i nduct i on in this setting. Recent in vitro studies with lysophosphatidylcholine, a component of oxi- dized low densi t y l i poprot ei n (and [3-very low density lipoprotein, anot her at herogeni c lipopro- tein in t he rabbit), have shown t hat this mat eri al can selectively up-regul at e VCAM-1 expression in cul t ured rabbit aortic endot hel i al cel l sY Conceiv- ably, this phospholipid may act al one or in combina- tion with cytokines gener at ed locally by activated vessel wall cells or emi grat i ng leukocytes, to up- regulate ELAM and thus cont ri but e to localized mononucl ear l eukocyt e recrui t ment . In addition to a VCAM- 1/ VLA- 4 adhesive inter- action, ot her endot hel i um- dependent count er - recept or mechani sms are pot ent i al l y rel evant to mononucl ear r ecr ui t ment in at herogenesi s. These include: i nt ercel l ul ar adhesi on mol ecul e- l , a widely expressed member of t he i mmunogl obul i n super- family that is also up-regul at ed in vascular endot he- lium as a chroni c ELAM and can i nt eract with various component s of t he CD11/ CD18 integrin complexl5; E-sel ect i n (ELAM-1), an acut e ELAM that interacts with sialyl-Lewis x and rel at ed carbo- hydrate ligands, 26 and an inducible endot hel i al count errecept or for L-selectin, a ligand constitu- tively present on blood l eukocyt e sur f aces. 27 Int er- estingly, al t hough all of t hese count er r ecept or pairs have been i mpl i cat ed in monocyt e- endot he- lium adhesi on, only VCAM- 1/ VLA- 4 has t he po- t ent i al to medi at e mononucl ear, l eukocyt e, but not pol ymor phonucl ear leukocyte, interactions. Two ot her rel evant endot hel i al product s t hat are se- cr et ed following cytokine activation include mono- cyte chemoat t r act ant pr ot ei n- l , a monocyt e-se- l ect ed chemoat t r act ant t hat has been i mpl i cat ed in monocyt e- endot hel i um transmigration, 28 and mac- rophage colony-stimulating factor, a cytokine t hat can pr omot e activation and mat ur at i on, of mono- cytes and macrophages. 29 Recent i mmunohi st o- chemi cal studies have localized t he expression of t hese mol ecul es at various stages of at heroscl erot i c lesion devel opment in animals and, in some in- stances, in humans. 3-32 The relative cont ri but i ons of t hese mechani sms of l eukocyt e r ecr ui t ment to t he at herogeni c process is an i mport ant ar ea of ongoing study t hat has i mport ant pat hogenet i c and t her apeut i c implications. PATHOPHYSIOLOGIC I MPUCATI ONS AND I~l tEl ~l l ~k CUNI CAL APPLICATIONS OF ATHERO-ELAM The identification of an inducible mononucl ear l eukocyt e-sel ect i ve adhesi on mol ecul e t hat is ex- pressed in developing at heroscl erot i c lesions in a well-defined exper i ment al modei has several con- cept ual as well as practical implications. First, it provides evi dence t hat endot hel i al act i vat i on/ dysfunction occurs early in t he at heroscl erot i c process. It suggests t hat t he net bal ance of local pathophysiologic stimuli has elicited a pat t er n of response in t he endot hel i um t hat is mani fest ed by this change in surface phenot ype. Second, t he i nt eract i on of endot hel i al VCAM-1 with its leuko- cyte count er r ecept or , VLA-4, is a mol ecul ar event in mononucl ear l eukocyt e r ecr ui t ment t hat may be susceptible to some form of ant i adhesi on t her apeu- tic intervention. Thi rd, charact eri zat i on of t he expression of VCAM-1 or ot her ATHERO- ELAM in human at heroscl erot i c lesions may provide novel mar ker s for t he early stages of this complex disease process. Clinical applications of such markers might i ncl ude noninvasive imaging of early lesions via l abel ed monocl onal antibodies, differential analy- sis of t he pat t er n of endot hel i al activation antigens as an i ndi cat or of t he stage of underl yi ng vessel wall disease, or selective t arget i ng to early at hero- sclerotic lesions of a conventional t herapeut i c agent or specifically engi neer ed genet i c const ruct for "gene t her apy. " Clearly, t he successful application of such diagnostic and t her apeut i c strategies will requi re bet t er under st andi ng of t he basic mecha- A SYMPOSIUM: ATHEROTHROMBOSIS 698 nisms of endot hel i al activation in t he cont ext o f human at heroscl erot i c vascular di sease. VASCULAR ENDOTHELIUM I S AN INTEGRATOR OF THE LOCAL PATHOPHYSIOLOGIC MI UEU As t he previ ous exampl es illustrate, t he endot he- lial lining of t he cardiovascular system is a dynami- cally mut abl e i nterface that can exhi bi t a spectrum o f adapti ve changes. Indeed, s ome o f its vast repertoi re o f autacoi ds, growth factors, and vasoac- tive, hemost at i c, and fibrinolytic substances of t e n contri bute to agoni st / ant agoni st bal ances that have i mportant i mpl i cati ons f or t he f unct i on o f t he vascular lining, adjacent vascular cells, and interact- ing bl ood const i t uent s. By vi rtue o f its uni que anatomi c posi ti on, t he endot hel i um also plays an i mportant role in t he local transduction and integra- tion o f di verse bi ol ogi c stimuli, i ncl udi ng circulat- ing hormones and bacterial products, l ocal l y gener- ated cytoki nes, and even bi omechani cal forces. Thus, in an i mportant sense, t he phenot ype o f an endot hel i al cell is a ref l ect i on o f t he local pat ho- physi ol ogi c mi l i eu. As our knowl edge o f t he stimuli and cons equences o f dysfuncti onal endot hel i al phe- not ypes i ncreases, so will our worki ng concept s o f t he pat hogenesi s o f atheroscl erosi s. We hope this will provi de a rati onal basis f or i nnovati ve di agnos- tic and t herapeut i c i nterventi ons in human coro- nary artery di sease in t he near future. Ac k n o wl e d g me n t s : The author wi shes to ac- knowl edge his col l eagues and col l aborators in t he experi ment al studi es summari zed here, especi al l y Drs. Tucker Col l i ns, Myron Cybulsky, Nori aki Kume, and Wi l l i am Luscinskas. REFERENCES 1. Virchow R. Der ateromatose Prozess der Arterien. VC'ten Med Wochenschr 1856i6:825-841. 2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1993;362:801-809. 3, Gimbrone MA Jr, ed. 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Exam Bio-Organic Chemistry (8S140) Thursday November 23, 2008 14.00-17.00 H This Exam Consists of 7 Questions. Explain Your Answers Clearly. Answers May Be Given in English or Dutch