Circulation Research. 2000;87:264-267
(Circulation Research. 2000;87:264.)
© 2000 American Heart Association, Inc.
Local Delivery of Ceramide for Restenosis
Is There a Future for Lipid Therapy?
Frank D. Kolodgie,
Andrew Farb,
Renu Virmani
From the Department of Cardiovascular Pathology, Armed Forces Institute
of Pathology, Washington, DC.
Correspondence to Renu Virmani, MD, Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, 6825 16th St NW, Washington, DC 20306-6000. E-mail virmani{at}afip.osd.mil
Key Words: ceramide restenosis angioplasty pathological remodeling
 |
Introduction
|
|---|
Despite the clinical advantage of
percutaneous transluminal
coronary angioplasty
(PTCA) in treating severely narrowed blood
vessels, long-term success
is often compromised by restenosis.
It is unclear what
mechanisms cause vessels to renarrow, despite
numerous studies
involving patients and experimental animal
models of
arterial injury. Although neointimal thickening
was
initially considered the major cause, recent evidence suggests
that
arterial constriction, adventitial thickening, or both
may
be critical in the restenosis process. The geometric changes
of
vessel expansion and contraction constitute the definition
of
remodeling. The present emphasis on remodeling and
restenosis
has been driven by studies failing to show a direct
correlation
between neointimal thickening and lumen size,
suggesting that
intimal mass alone is insufficient to explain
narrowing.
1 Analyses
of human coronary
angioplasty sites at autopsy by our laboratory
support the thesis that
constrictive remodeling and the initial
plaque burden, rather than
neointimal formation, are responsible
for the failure of
angioplasty.
2 Other potential contributors
to the
pathophysiology of restenosis include arterial
spasm,
vessel recoil, platelet aggregation, and thrombus
formation.
Arterial injury evokes a sequence of events, consisting of
medial smooth muscle cell activation, migration and proliferation, and
matrix secretion culminating in a thickened
neointima.3 This series of events is linked to
complex interactions between cells within the vessel wall as well as
circulating blood cells and cytokines. At least in theory,
restenosis should be a treatable process with adjunctive
pharmacotherapy. Several agents, such as vascular
endothelial growth factor,4
heparin,5 paclitaxel,6 7
urokinase,8 recombinant hirudin,9
colchicine,10 11 rapamycin sirolimus,12 13
cytochalasin B,14 IIb and IIIa
inhibitors,15 and antisense
oligomers,16 17 18 19 have been tested in animal models or
patients. Most of these agents have failed to solve the
restenosis problem in humans, but two recent promising agents,
paclitaxel and rapamycin, are in clinical trials in Europe.
Several factors may contribute to the failure of studies to be
translated into successful human trials. Difficulties in achieving the
appropriate dose may offer one explanation. This can be remedied by
localized delivery of a drug rather than systemic administration, which
should help attain high concentrations while limiting the side effects
of potentially toxic agents. Alternatively, most of these agents have
generally targeted the reduction of tissue growth as a means of
suppressing the intimal hyperplasia, with little emphasis on vascular
remodeling, a factor perhaps more critical to the restenotic
process, at least in PTCA. Moreover, the importance of cell
proliferation in human restenosis is not well supported;
coronary atherectomy specimens at various intervals after PTCA
fail to show clinical evidence of increased cell
proliferation.20 This observation, however, does not mean
that proliferation does not occur. Obviously, smooth muscle cells are
the dominant cellular element of restenotic lesions; hence, it
is conceivable that replication occurs for transient periods, which may
be overlooked because of insufficient sampling. The extent to which
smooth muscle cell migration contributes to the restenotic
lesion must also be weighed.
Another point to consider is the course of the disease. Animal models
of restenosis generally consist of nonatherosclerotic vessels
studied within weeks of injury; in humans, this process typically
develops over 6 months. Finally, in reference to the underlying
disease, there is little information as to the influence of plaque
substrate on restenosis. In other words, animal models without
underlying atherosclerotic disease may be overly simplistic. For
example, it is conceivable that plaques with a large lipid core and
increased inflammation provide a more reactive environment than
relatively acellular fibrous plaques with small or absent lipid cores.
Preliminary evidence in support of this hypothesis comes from an
autopsy study of human stents by Farb et al,21
demonstrating that lipid core size and inflammation correlate with
neointima formation and greater restenosis. Today,
in-stent restenosis has become an emerging clinical problem,
because
80% of coronary patients receive stents rather than
PTCA alone.22
In a study published in this issue of Circulation Research,
Charles et al23 show that local delivery of
C6-ceramide, a cell-permeable bioactive analog of
ceramide, reduces neointimal hyperplasia by 50% 2 weeks
after balloon stretch injury of the rabbit carotid
artery.23 When neointimal stenosis was
quantified as a ratio of neointimal and medial
cross-sectional areas, ceramide treatment resulted in a 92% reduction
in stenosis. Although the growth-arresting potential of
ceramide has been shown previously in culture, this is the first study
demonstrating efficacy in an in vitro animal model of
restenosis. Administration of ceramide resulted in an early
inhibition of extracellular signalrelated kinase (ERK) and
phosphorylation of protein kinase B (PKB/Akt) as a
mechanism of decreased neointimal formation. The
downregulation of these activities highlights their importance in the
mitogenic and cell-survival pathways critical to
neointimal hyperplasia in animal models of
restenosis. Activation of PKB/Akt and ERK may represent
common signaling pathways of vascular smooth muscle cell migration and
growth as shared by several agonists, as has been shown recently with
angiotensin II.24 25 Pathological
analysis demonstrated that ceramide treatment did not provoke
inflammation, a desirable property for an antirestenotic drug.
As an important practical application, this study should prompt trials
in larger animal models of restenosis, including those that
involve stents. In addition, the development of ceramide-like analogs
with greater potency or long-term retention may also prove
beneficial.
 |
Ceramide: Metabolism and Catabolism
|
|---|
Ceramide forms the central molecule of all
sphingolipids.
26 27 Sphingolipids consist of a long-chain
amino-dialcohol base
(sphingoid), an amide-linked fatty acyl group, and
a polar or
glycosidic head group. Although these molecules were
initially
characterized as serving a structural role in membranes,
various
derivatives are recognized as second messengers that possess
diverse
cellular responses. Endogenous ceramide is
generated through
the activation of distinct sphingomyelinases residing
in separate
subcellular compartments in response to specific stimuli.
Ceramidases
are enzymes that hydrolyze ceramide to liberate the fatty
acid
from the sphingoid base to form sphingosine. The sphingosine
released
by ceramidases can be phosphorylated by
sphingosine kinase to
sphingosine-1-phosphate, which is also
biologically active in
vascular cells. In addition to the parent
ceramide, there are
cell-permeable analogs, such as
C
2-ceramide (
D-erythro-sphingosine,
N-acetyl),
C
6-ceramide
(
D-erythro-sphingosine,
N-hexanoyl),
or C
8-ceramide
(
D-erythro-sphingosine,
N-octanoly).
These exogenous cell-permeant ceramide analogs
mimic many of the
biological effects of natural ceramide.
It is apparent from the literature that the specificity of cellular
responses to ceramide depends on many factors, which include the nature
of the stimulus, costimulatory signals, and the cell type involved.
This diversity can result in the varied effects of ceramide on cell
function, some of which may be proatherogenic.26 27
Therefore, differential effects of ceramide may be observed, depending
on the dosing and extent of
hypercholesterolemia.
 |
Healing and Restenosis
|
|---|
Arterial injury produces a wound-healing response
initiated
by platelet deposition followed by inflammation,
angiogenesis,
granulation tissue, migration and proliferation of smooth
muscle
cells, and synthesis of matrix molecules. In the study by
Charles
et al,
23 neointimal formation was
studied at 2 weeks after
arterial injury, a time at which
healing may be incomplete,
especially with drug treatment. It is
unknown whether the beneficial
effects of ceramide on
restenosis represent a true reduction
in
neointima or delayed healing. Pathological examination was
restricted
to hematoxylin and eosinstained slides; special stains
were
not done for fibrin, for example, which in some instances can
be
mistaken for collagen.
28
Other antiproliferative drugs, such as paclitaxel, a novel
antimicrotubular agent with antiproliferative as well as antimigratory
properties, decreases neointimal formation 28 days after
stenting of rabbit iliac arteries. However, this effect is accompanied
by incomplete healing up to 6 months after stenting.29 In
the study by Charles et al,23 inflammation was assessed at
only up to 60 minutes after injury; the inflammatory response in this
model peaks between 3 to 5 days.23 It has been shown that
ceramides mimic the effects of cytokines, which can induce
cellular inflammation via activation of stress-activated
protein kinase cascades.30 Therefore, more detailed
pathological studies should be carried out to determine the long-term
effects of ceramide treatment on inflammation and healing in vivo.
The dosing of ceramide also deserves comment. In the report by Charles
et al,23 the signaling effects of ceramide were apparent
as early as 15 minutes after dosing. This was accompanied by a 50%
loss of the parent ceramide analog by 60 minutes after localized
delivery. Curiously, the decrease in ceramide mass was associated with
an increase in gangliosides and cerebrosides but not sphingosines;
phosphorylated sphingosine is a biologically active
metabolite associated with prothrombogenic effects.27
Whether this short half-life of ceramide is enough to sustain the wave
of cellular events in the presence of an injured artery with
atherosclerotic disease is debatable. Moreover, the ceramide content of
atherosclerotic lesions is markedly elevated relative to normal
arteries, in part from the LDL content. The ceramide concentration of
lesional LDL is increased 10- to 50-fold compared with plasma
LDL.31 From these data, one would assume that the ceramide
levels would be greater in a lipid-rich plaque relative to a more
fibrous lesion. This poses the question of whether localized delivery
of exogenous ceramide to a plaque abundant in natural ceramide would
have any additional effects. Finally, localized delivery of
radiolabeled ceramide was observed throughout all layers of the
arterial media. It remains to be determined how well
exogenous ceramide can penetrate a well-collagenized atherosclerotic
plaque.
The model of rabbit balloon injury of normal vessels used by Charles et
al23 produces a neointima consisting of a
relatively homogeneous population of smooth muscle cells.
The effects of ceramide in a restenosis model with underlying
atherosclerotic disease need to be explored. The ceramide-induced
decrease in cell proliferation was measured using an antibody directed
against proliferating cell nuclear antigen (PCNA). It is recognized
that PCNA is a protein that is required for both DNA replication and
DNA repair32 ; positive PCNA staining may have
represented cellular injury. PCNA is also upregulated
during apoptosis, because DNA strand breaks induce an
ultimately futile DNA repair process.33 This effect is
probably of little importance in the carotid stretch-injury model,
because an increase in apoptosis was not associated with either
control or ceramide-treated animals.
 |
Ceramide and Intracellular Signaling
|
|---|
The diversity of ceramide as a messenger of cell signaling is
well
established. Not only can ceramide alone serve as a second
messenger in
response to a variety of stimuli, but it may also
be converted to other
structural or effector molecules. Obviously,
ceramide signaling is a
nascent area of vascular research, and
many of its actions need to be
identified. In the article by
Charles et al,
23 ceramide is
a potent inhibitor of PKB/Akt
and ERK signaling in vascular
smooth muscle. Inhibition of PKB/Akt
by ceramide has been recently
shown to be mediated by dephosphorylation
of serine
473.
34 Moreover, an apparent crosstalk between both
PKB/Ark
and ERK pathways has also been recognized.
35 Other
pathways
of ceramide signaling may involve inhibition of
Ca
2+-activated
K
+
channels or protein kinase C, both promoters of vasodilation.
Ceramide
can also mimic the effects of certain cytokines, such
as tumor
necrosis factor-

, causing activation of other regulatory
mediators,
including nuclear factor-

B, activator protein-1,
and
c-Jun N-terminal kinase.
36 Finally, the cytotoxicity
associated
with ceramide has been linked to its effects on
mitochondrial
membrane permeability and downstream activation of
caspases.
37
 |
Summary
|
|---|
How a structurally simple molecule like ceramide is able to
mediate
so many different, and sometimes paradoxical,
physiological
responses ranging from cell
proliferation and differentiation
to inhibition of cell growth and
apoptosis is unknown. Moreover,
crosstalk between
ceramide-induced signal transduction cascades
and other signaling
pathways will add to the inherent difficulty
in distinguishing the
specific effects of ceramide in vascular
biology. Although the study by
Charles et al
23 shows promise,
this is the first step
toward our understanding of the actions
of ceramide on
restenosis in vivo. Clearly, some of the vascular
targets of
ceramide could be clarified through studies using
animals harboring
disrupted genes of sphingolipid metabolism.
More extensive
research on the interaction of ceramide with
specific cell types,
especially in more complex models of restenosis
using stents,
is needed. More importantly, a clearer understanding
of how vessels
renarrow, particularly with stents, may help
decipher the signaling
pathways that promote restenosis; only
then can the development
of new therapeutic strategies be clinically
effective.
 |
Footnotes
|
|---|
The opinions expressed in this editorial are not necessarily
those of the editors or of the American Heart Association.
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting views of the Departments of the Army, Air Force, or Defense.
 |
References
|
|---|
-
Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler
LF, Wong C, Hong MK, Kovach JA, Leon MB. Arterial
remodeling after coronary angioplasty: a serial intravascular
ultrasound study. Circulation. 1996;94:3543.[Abstract/Free Full Text]
-
Sangiorgi G, Taylor AJ, Farb A, Carter AJ, Edwards WD,
Holmes DR, Schwartz RS, Virmani R. Histopathology of
postpercutaneous transluminal coronary
angioplasty remodeling in human coronary arteries. Am
Heart J. 1999;138:681687.[Medline]
[Order article via Infotrieve]
-
Schwartz SM. Smooth muscle migration in
atherosclerosis and restenosis. J
Clin Invest. 1997;100:S87S89.
-
Asahara T, Bauters C, Pastore C, Kearney M, Rossow S,
Bunting S, Ferrara N, Symes JF, Isner JM. Local delivery of vascular
endothelial growth factor accelerates
reendothelialization and attenuates intimal hyperplasia
in balloon-injured rat carotid artery. Circulation. 1995;91:27932801.[Abstract/Free Full Text]
-
Lablanche JM, McFadden EP, Meneveau N, Lusson JR,
Bertrand B, Metzger JP, Legrand V, Grollier G, Macaya C, de Bruyne B,
Vahanian A, Grentzinger A, Masquet C, Wolf JE, Tobelem G, Fontecave S,
Vacheron A, dAzemar P, Bertrand ME. Effect of nadroparin, a
low-molecular-weight heparin, on clinical and angiographic
restenosis after coronary balloon angioplasty: the FACT
study. Fraxiparine Angioplastie Coronaire Transluminale.
Circulation. 1997;96:33963402.[Abstract/Free Full Text]
-
Sollott SJ, Cheng L, Pauly RR, Jenkins GM, Monticone
RE, Kuzuya M, Froehlich JP, Crow MT, Lakatta EG, Rowinsky EK, Kinsella
JL. Taxol inhibits neointimal smooth muscle cell
accumulation after angioplasty in the rat. J Clin
Invest. 1995;95:18691876.
-
Herdeg C, Oberhoff M, Baumbach A, Blattner A, Axel DI,
Schroder S, Heinle H, Karsch KR. Local paclitaxel delivery for the
prevention of restenosis: biological effects and efficacy in
vivo. J Am Coll Cardiol. 2000;35:19691976.[Abstract/Free Full Text]
-
Wilensky RL, Pyles JM, Fineberg N. Increased thrombin
activity correlates with increased ischemic event rate after
percutaneous transluminal coronary angioplasty:
lack of efficacy of locally delivered urokinase. Am Heart
J. 1999;138:319325.[Medline]
[Order article via Infotrieve]
-
Herrman JP, Simon R, Umans VA, Peerboom PF, Keane D,
Rijnierse JJ, Bach D, Kobi P, Kerry R, Close P, Deckers JW, Serruys PW.
Evaluation of recombinant hirudin (CGP 39,393/TMREVASC) in the
prevention of restenosis after percutaneous
transluminal coronary angioplasty: rationale and design of the
HELVETICA trial, a multicentre randomized double blind heparin
controlled study. Eur Heart J. 1995;16(suppl
L):5662.
-
Gradus-Pizlo I, Wilensky RL, March KL, Fineberg N,
Michaels M, Sandusky GE, Hathaway DR. Local delivery of biodegradable
microparticles containing colchicine or a colchicine analogue: effects
on restenosis and implications for catheter-based drug
delivery. J Am Coll Cardiol. 1995;26:15491557.[Abstract]
-
OKeefe JH Jr, McCallister BD, Bateman TM, Kuhnlein
DL, Ligon RW, Hartzler GO. Ineffectiveness of colchicine for the
prevention of restenosis after coronary angioplasty.
J Am Coll Cardiol. 1992;19:15971600.[Abstract]
-
Gallo R, Padurean A, Jayaraman T, Marx S, Roque M,
Adelman S, Chesebro J, Fallon J, Fuster V, Marks A, Badimon JJ.
Inhibition of intimal thickening after balloon angioplasty in porcine
coronary arteries by targeting regulators of the cell cycle.
Circulation. 1999;99:21642170.[Abstract/Free Full Text]
-
Carter AJ, Bailey LR, Llanos G, Lieuallen W, Kopia G,
Papandreou G, Narayan P, Falotico R, Adelman S, Leon MB. Stent based
sirolimus delivery reduces neointimal proliferation in a
porcine coronary model of restenosis. J Am
Coll Cardiol. 2000;35(suppl A):13A. Abstract.
-
Lehmann KG, Popma JJ, Werner JA, Lansky AJ, Wilensky
RL. Vascular remodeling and the local delivery of cytochalasin B after
coronary angioplasty in humans. J Am Coll
Cardiol. 2000;35:583591.[Abstract/Free Full Text]
-
Lefkovits J, Ivanhoe RJ, Califf RM, Bergelson BA,
Anderson KM, Stoner GL, Weisman HF, Topol EJ. Effects of platelet
glycoprotein IIb/IIIa receptor blockade by a chimeric
monoclonal antibody (abciximab) on acute and six-month outcomes after
percutaneous transluminal coronary angioplasty
for acute myocardial infarction: EPIC investigators. Am J
Cardiol. 1996;77:10451051.[Medline]
[Order article via Infotrieve]
-
Zhu NL, Wu L, Liu PX, Gordon EM, Anderson WF, Starnes
VA, Hall FL. Downregulation of cyclin G1 expression by
retrovirus-mediated antisense gene transfer inhibits vascular smooth
muscle cell proliferation and neointima formation.
Circulation. 1997;96:628635.[Abstract/Free Full Text]
-
Robinson KA, Chronos NA, Schieffer E, Palmer SJ,
Cipolla GD, Milner PG, King SB III. Endoluminal local delivery of
PCNA/cdc2 antisense oligonucleotides by porous balloon
catheter does not affect neointima formation or vessel size
in the pig coronary artery model of postangioplasty
restenosis. Cathet Cardiovasc Diagn. 1997;41:348353.[Medline]
[Order article via Infotrieve]
-
Hanna AK, Fox JC, Neschis DG, Safford SD, Swain JL,
Golden MA. Antisense basic fibroblast growth factor gene transfer
reduces neointimal thickening after arterial
injury. J Vasc Surg. 1997;25:320325.[Medline]
[Order article via Infotrieve]
-
Pickering JG, Isner JM, Ford CM, Weir L, Lazarovits A,
Rocnik EF, Chow LH. Processing of chimeric antisense
oligonucleotides by human vascular smooth muscle cells
and human atherosclerotic plaque: implications for antisense therapy of
restenosis after angioplasty. Circulation. 1996;93:772780.[Abstract/Free Full Text]
-
OBrien ER, Alpers CE, Stewart DK, Ferguson M, Tran N,
Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM.
Proliferation in primary and restenotic coronary
atherectomy tissue: implications for antiproliferative therapy.
Circ Res. 1993;73:223231.[Abstract/Free Full Text]
-
Farb A, Weber DK, Jones R, Virmani R. Plaque substrate
and arterial damage are predictors of
restenosis after coronary stenting in humans.
J Am Coll Cardiol. 2000;35(suppl A):4A. Abstract.
-
Grines CL, Cox DA, Stone GW, Garcia E, Mattos LA,
Giambartolomei A, Brodie BR, Madonna O, Eijgelshoven M, Lansky AJ,
ONeill WW, Morice MC. Coronary angioplasty with or without
stent implantation for acute myocardial infarction: stent primary
angioplasty in Myocardial Infarction Study Group. N Engl
J Med. 1999;341:19491956.[Abstract/Free Full Text]
-
Charles R, Sandirasegarane L, Yun J, Bourbon N, Wilson
R, Rothstein RP, Levison SW, Kester M. Ceramide-coated balloon
catheters limit neointimal hyperplasia after stretch injury
in carotid arteries. Circ Res. 2000;87:282288.[Abstract/Free Full Text]
-
Xi XP, Graf K, Goetze S, Fleck E, Hsueh WA, Law RE.
Central role of the MAPK pathway in Ang IImediated DNA synthesis and
migration in rat vascular smooth muscle cells. Arterioscler
Thromb Vasc Biol. 1999;19:7382.[Abstract/Free Full Text]
-
Hattori Y, Akimoto K, Kasai K. The effects of
thiazolidinediones on vascular smooth muscle cell activation by
angiotensin II. Biochem Biophys Res Commun. 2000;273:11441149.[Medline]
[Order article via Infotrieve]
-
Chatterjee S. Sphingolipids in
atherosclerosis and vascular biology.
Arterioscler Thromb Vasc Biol. 1998;18:15231533.[Abstract/Free Full Text]
-
Auge N, Negre-Salvayre A, Salvayre R, Levade T.
Sphingomyelin metabolites in vascular cell signaling and atherogenesis.
Prog Lipid Res. 2000;39:207229.[Medline]
[Order article via Infotrieve]
-
Taylor AJ, Gorman PD, Farb A, Hoopes TG, Virmani R.
Long-term coronary vascular response to
32P ß-particleemitting stents in a canine
model. Circulation. 1999;100:23662372.[Abstract/Free Full Text]
-
Drachman DE, Edelman ER, Kamath KR, Palasis M, Yang D,
Nott SH, Rogers C. Sustained stent-based delivery of paclitaxel arrests
neointimal thickening and cell proliferation.
Circulation. 1998;98(suppl I):I-740. Abstract.
-
Coroneos E, Wang Y, Panuska JR, Templeton DJ, Kester M.
Sphingolipid metabolites differentially regulate extracellular
signal-regulated kinase and stress-activated protein kinase
cascades. Biochem J. 1996;316:1317.
-
Schissel SL, Jiang X, Tweedie-Hardman J, Jeong T,
Camejo EH, Najib J, Rapp JH, Williams KJ, Tabas I. Secretory
sphingomyelinase, a product of the acid sphingomyelinase gene, can
hydrolyze atherogenic lipoproteins at neutral pH: implications for
atherosclerotic lesion development. J Biol Chem. 1998;273:27382746.[Abstract/Free Full Text]
-
Savio M, Stivala LA, Bianchi L, Vannini V, Prosperi E.
Involvement of the proliferating cell nuclear antigen (PCNA) in DNA
repair induced by alkylating agents and oxidative damage in human
fibroblasts. Carcinogenesis. 1998;19:591596.[Abstract/Free Full Text]
-
Berges RR, Furuya Y, Remington L, English HF, Jacks T,
Isaacs JT. Cell proliferation, DNA repair, and p53 function are not
required for programmed death of prostatic glandular cells induced by
androgen ablation. Proc Natl Acad Sci U S A. 1993;90:89108914.[Abstract/Free Full Text]
-
Schubert KM, Scheid MP, Duronio V. Ceramide inhibits
protein kinase B/Akt by promoting dephosphorylation of
serine 473. J Biol Chem. 2000;275:1333013335.[Abstract/Free Full Text]
-
Dimmeler S, Zeiher AM. Akt takes center stage in
angiogenesis signaling. Circ Res. 2000;86:45.[Free Full Text]
-
Manna SK, Sah NK, Aggarwal BB. Protein tyrosine kinase
p56lck is required for ceramide-induced but not tumor necrosis
factor-induced activation of NF-
B, AP-1, JNK, and
apoptosis. J Biol Chem. 2000;275:1329713306.[Abstract/Free Full Text]
-
Jacotot E, Costantini P, Laboureau E, Zamzami N, Susin
SA, Kroemer G. Mitochondrial membrane permeabilization during the
apoptotic process. Ann N Y Acad Sci. 1999;887:1830.[Abstract/Free Full Text]