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Vascul ar Endothel i um: An I nt egr at or of

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.
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.
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
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-
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.
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-
nisms of endot hel i al activation in t he cont ext o f
human at heroscl erot i c vascular di sease.
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.
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