Geshi, Eiichi

Iso, Yoshitaka

Katagoro, Takashi

Koba, Shinji

Sato, Takatoshi

Shoji, Makoto

Soda, Teruko

Suzuki, Hiroshi

Tsunoda, Fumitaka

Elsevier - Publisher Connector 

1064-178 A novel oral neutrophil elastase inhibitor, ONO-6818, suppressed early atherosclerotic plaque formation in apolipoprotein E-knockout mice 

JACC March 3, 2004 ABSTRACTS - Vascular Disease, Hypertension, and Prevention  465A
Vascular Disease, Hypertension, and Prevention
Conclusions: The ratio of OxLDL to HDL seems to be an important predictor of subse-
quent MI in patients with UCAD.
POSTER SESSION
1064 
Pathogenesis and Treatment of the 
Atherosclerotic Plaque
Monday, March 08, 2004, 9:00 a.m.-11:00 a.m.
Morial Convention Center, Hall G
Presentation Hour: 9:00 a.m.-10:00 a.m.
1064-178 A Novel Oral Neutrophil Elastase Inhibitor, ONO-6818, 
Suppressed Early Atherosclerotic Plaque Formation in 
Apolipoprotein E-Knockout Mice
Yoshitaka Iso, Hiroshi Suzuki, Teruko Soda, Fumitaka Tsunoda, Takatoshi Sato, Makoto 
Shoji, Shinji Koba, Eiichi Geshi, Takashi Katagoro, Showa University School of Medicine, 
Tokyo, Japan
Objective: Proteolysis and elastolysis contribute to the development of atherosclerosis.
Neutrophil elastase (NE) can degrade not only elastin but also collagens, fibronectin, and
proteoglycans, and it is predominantly present in neutrophils. However, recent studies
indicated the expression of NE in atheroma-related cells. Therefore, we hypothesized NE
inhibition prevented atherogenesis, and examined whether ONO-6818, an orally active
inhibitor of NE, limited atherosclerotic lesion in apolipoprotein E-knockout mice (ApoE-
KO mice). Methods and Results: ApoE-KO mice (8 weeks of age) were fed a normal
chow diet, and euthanized at the age of 12 weeks. They were divided into 2 groups; (1)
control group (n=8, oral administration of saline) (2) treatment group (n=8, oral adminis-
tration of 100mg/kg ONO-6818 for 4weeks). Plasma cholesterol levels were not signifi-
cantly different between the two groups at the age of 12 weeks. Histological analysis of
atherosclerotic lesions in the aortic root was performed using oil red O stained-sections.
Plaque area was significantly reduced in ApoE-KO mice treated with ONO-6818 com-
pared to the control group (9377 +/- 2078 µm2 versus 28464 +/- 7613 µm2, p=0.02). In
immunohistochemical study, the percent area of macrophage accumulation to the total
cross-sectional vessel wall area was smaller in ApoE-KO mice treated with ONO-6818
than the control group (1.0 +/- 0.2% versus 2.8 +/- 0.9%, p=0.07). The abdominal aortas
of ApoE-KO mice were used for RT-PCR analysis of eNOS mRNA. ONO-6818 prevented
downregulation of eNOS mRNA expression in ApoE-KO mice compared to the controls.
Conclusions: NE inhibitor suppressed early atherosclerotic plaque formation and pro-
tected endothelial function in ApoE-KO mice. These results suggested that NE could be a
new therapeutic target for atherogenesis.
1064-179 Mice Deficient in Leptin or the Leptin Receptor Exhibit 
Reduced Neointima Formation Following Vascular 
Injury
Yuechun Shen, Peter F. Bodary, Daniel T. Eitzman, University of Michigan, Ann Arbor, MI
Background: Elevated levels of plasma leptin have been identified as an independent
risk factor for cardiovascular disease, although the mechanism by which leptin increases
this risk is unknown. We hypothesize that leptin may contribute to vascular disease
through effects on vascular remodeling.
Methods and results: A mouse model of femoral arterial wire injury was used that
induces endothelial damage followed by intimal hyperplasia. We induced arterial injury in
wild type (Lep+/+) (n=8), leptin deficient (Lep-/-) (n=13) and leptin receptor deficient
(LepR-/-) (n=9) mice. Four weeks after injury, the intima/media ratios (I/Ms) of Lep-/- and
LepR-/- mice were 0.80±0.14 and 0.55±0.20, which were significantly reduced compared
to Lep+/+ mice (1.50±0.22) (p<0.01 and <0.02, respectively). To further establish that
leptin was responsible for the altered neointima formation, leptin was replaced in Lep-/-
mice by daily peritoneal injection beginning one day prior to injury until sacrifice. The I/Ms
of Lep-/- mice (n=8) receiving leptin treatment were significantly higher (1.44±0.18) than
Lep-/- mice described in the original cohort (p<0.02). Mice were also treated with an ade-
novirus, expressing murine leptin on the RSV promoter, one day following injury. The I/Ms
of Lep-/- mice (n=6) receiving ad-leptin were 2.04±0.27, which were significantly higher
compared to the I/Ms of the control Lep-/- mice (p<0.004). The I/Ms of LepR-/- mice (n=4)
receiving ad-leptin were 0.50±0.19, which were not different from the I/Ms in the original
group of LepR-/- mice.
Conclusion: These observations demonstrate that mice lacking leptin or the leptin
receptor are protected from neointima formation following vascular injury and suggest
that leptin may play a role in atherosclerosis by promoting lesion growth.
1064-180 Splenectomy and Adoptive Transfer of Splenocytes 
Reveal a Critical Role for Spleen in Mediating Athero-
Protective Effects of Immunization With Apolipoprotein 
B-100-Related Peptide Sequence in Apo E (-/-) Mice
Kuang-Yuh Chyu, Stephanie M. Babbidge, Paul C. Dimayuga, Juliana Yano, Jan Nilsson, 
Bojan Cercek, Prediman K. Shah, Gunilla Nordin Fredrikson, Cedars-Sinai Medical 
Center, Los Angeles, CA, Lund University, Malmo, Sweden
Background: We have previously reported athero-protective effects of immunizing
hypercholesterolemic apo E (-/-) mice with single or mixture of human apo B 100 related
peptides. One such peptide is peptide 2 (ATRFKHLRKYTYNYEAESSS, 95% homology
with murine apo B 100). In this study, we sought to determine the role of spleen in medi-
ating athero-protective effects of peptide 2 immunization.
Methods and Results: Male apo E (-/-) mice were immunized with 33 mcg of peptide 2
at 6 weeks of age and boosted at 9 weeks with Alum as adjuvant. Control group received
Alum only. At 12 weeks splenocytes were harvested, pooled and injected into non-immu-
nized apo E (-/-) mice via tail vein. Both donor and recipient groups were fed high choles-
terol diet until sacrifice at 25 weeks. Splenectomy abolished athero-protective effect of
peptide 2 immunization; whereas adoptive transfer of splenocytes from peptide 2 immu-
nized mice reduced atherosclerosis and cholesterol levels in recipient unimmunized
mice. (Table)
Conclusion: Our data provide proof of principle that spleen plays an important role in
mediating the atheroprotective effects of immunization with a single apo B 100 related
peptide in apo E (-/-) mice. 
When compared to Alum recipient group “*” p < 0.05 and “#” p=0.07 by t-test
1064-181 Distinct Signaling Pathways Mediate Protease Activated 
Receptor-Dependent Endothelial Exocytosis
John H. Cleator, Douglas E. Vaughan, Heidi E. Hamm, Vanderbilt University, Nashville, 
TN
Thrombin stimulation of the release von Willebrand Factor (vWF) and P-selectin from
endothelial cells results in propagation of thrombotic events (vWF- mediated platelet
adhesion and aggregation) and adhesion of neutrophils through P-selectin.The purpose
of this study is to determine which Protease Activated Receptors are responsible for
mediating thrombin-induced release of vWF and P-selectin from human umbilical vein
endothelial cells (HUVEC). HUVEC were stimulated with thrombin, specific PAR-activat-
ing peptides (PAR-AP) or histamine and released vWF was measured in the media
(ELISA), while P-selectin was measured on the cell membranes using a cell-based
ELISA. PAR1-AP and PAR2-AP stimulation of vWF release was less effective when com-
pared to either thrombin or histamine stimulation. However, PAR1-AP stimulation of P-
selectin was as efficacious as histamine or thrombin stimulation. In contrast, PAR2-AP
was significantly less efficacious in stimulating the release of P-selectin than vWF. PAR2-
AP stimulation of mobilization of intracellular Ca2+ was nearly identical to PAR1-AP or
thrombin stimulation of mobilization of intracellular Ca2+. Calcium chelation with BAPTA
nearly completely inhibited thrombin-mediated release of vWF while only inhibiting 50-
60% of thrombin-mediated cell surface expression of P-selectin, suggesting an additional
Ca2+-independent pathway involved in release of P-selectin. Furthermore, pertussis toxin
inhibited the release of vWF, while not effecting P-selectin release, implicating Gi/o in
mediating the release of vWF, but not P-selectin. Immunostaining of P-selectin and vWF
demonstrated co-localization of the two proteins in characteristic Weibel-Palade bodies.
Differential signaling and co-localization of P-selectin with vWF suggest PARs stimulate
the differential release of distinct fractions of Weibel-Palade bodies. Selective pharmaco-
logical targeting of specific PARs and G proteins provides a novel therapeutic strategy for
altering the hemostatic and inflammatory balance of specific vascular beds.
1064-182 Gene Transfer of an ApoA-I Mimetic Peptide Reduces 
Atherosclerosis in Mice
Peter F. Bodary, Yuechun Shen, Randal J. Westrick, Narendra D. Lalwani, Sandra L. 
Drake, Jean-Louis H. Dasseux, Daniel T. Eitzman, University of Michigan, Ann Arbor, MI, 
Esperion Therapeutics, Inc., Ann Arbor, MI
Background. Apolipoprotein A-I (ApoA-I) has been shown to be protective against vas-
cular disease in animal models. The development of peptide-based mimetics of ApoA-1
may provide a particularly useful therapy for patients with atherosclerotic vascular dis-
ease. The goal of this study was to assess the effect of a novel ApoA-I peptide analogue
(ESP 24218, Esperion Therapeutics) designed to activate LCAT and promote reverse
lipid transport (RLT).
Methods and results. Nine week old male mice deficient in the LDL receptor were
begun on a western chow diet to accelerate atherogenesis. At 14 weeks of age, mice
were injected intravenously with saline (n=9) or a recombinant adenovirus expressing
either wild-type ApoA-I (n=9), a 22 amino acid ApoA-I peptide analogue, ESP 24218,
(n=10) or a 22 amino acid control peptide (n=10). At 18 weeks of age, mice were sacri-
ficed and the atherosclerotic surface area was quantified in the aortic arch. The amount
of atherosclerosis was significantly lower in mice expressing either wild-type ApoA-I
Death MI
OxLDL <median (n=206) 8 (4%) 19 (9%)
>median (n=226) 9 (4%) Ns 38 (17%) P=0.02
OxLDL/HDL <median (n=208) 9 (4%) 18 (9%)
>median (n=208) 6 (3%) Ns 37 (18%) P=0.006
LDL <median (n=189) 5 (3%) 26 (14%)
>median (n=197) 9 (5%) Ns 25 (13%) Ns
Cholesterol
(mg/dl)
Aortic plaque
(% area)
Aortic sinus 
plaque size 
(mm sq)
MOMA 
immunoreactivity 
(% plaque)
Alum donor, n=4 1285±114 5.6±5.3 0.55±0.05 4.3±4.6
P2 donor, n=9 910±459 3.0±1.7 0.45±0.18 4.2±3.5
Alum recipient, n=10 941±328 5.1±3.0 0.46±0.15 4.8±3.0
P2 recipient, n=9 560±239* 2.1±1.8* 0.33±0.13# 4.7±5.1


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1064-178 A novel oral neutrophil elastase inhibitor, ONO-6818, suppressed early atherosclerotic plaque formation in apolipoprotein E-knockout mice

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