Demidenko, Eugene

Friedman, Bruce J

Grinberg, Oleg Y

Grinberg, Stalina A

Hou, Huagang

Moodie, Karen L

Post, Mark J

Swartz, Harold M

Tomaszewski, Jane

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472A ABSTRACTS - Vascular Disease, Hypertension, and Prevention JACC  March 3, 2004
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1066-198 Incidence and Characteristics of Ruptured Plaque in 
Human Femoro-Popliteal Arteries
Yoichiro Hongo, Ali Hassan, Krishnankutty Sudhir, Paul G. Yock, Peter J. Fitzgerald, 
Yasuhiro Honda, Stanford University, Stanford, CA
Background: Numerous studies have reported the presence of plaque rupture (PR) at
the culprit lesions of acute coronary syndrome. However the incidence and clinical signif-
icance of PR in the peripheral circulation have not been well studied.
Methods: Sixty-six patients with intermittent claudication scheduled for elective angio-
plasty were enrolled per protocol, a femoro-popliteal segment containing one culprit
lesion with > 75% stenosis by angiography was selected for IVUS analysis (mean seg-
ment length: 11cm). IVUS was performed prior to any intervention. Discontinuity of lumi-
nal surface with a cavity within the plaque mass was defined as PR. Incidence and
location of PR as well as relationship to clinical risk profiles were evaluated.
Results: Fifty-four PR were observed in 35 (53%) out of 66 segments. Three segments
had 3 PR and 12 had 2 PR. Thirty-eight out of 54 PR were observed outside culprit
lesions. When patients with or without PR were compared, only incidence of previous
myocardial infarction (MI) was significantly higher in patients with PR. (Table)
Conclusion: In the femoro-popliteal circulation, the appearance of PR is common (53%).
Higher incidence of prior MI in patients with PR may be reflective of a more generalized
vulnerable plaque environment, i.e., “vulnerable patients” with pan-vascular inflamma-
tion.
ORAL CONTRIBUTIONS
805 
Advances in Angiogenesis
Monday, March 08, 2004, 9:15 a.m.-10:30 a.m.
Morial Convention Center, Room 265
9:15 a.m.
805-1 Indirect Imaging of Vascular Endothelial Growth Factor 
Gene Expression Using a Positron Emission 
Tomography Reporter Gene
Joseph C. Wu, Ian Y. Chen, Gobalakrishnan Sundaresan, Yanling Wang, Ronald G. 
Crystal, Sanjiv S. Gambhir, David Geffen UCLA School of Medicine, Los Angeles, CA, 
Weill Medical College of Cornell University, New York, NY
Background: Recent clinical trials with vascular endothelial growth factor (VEGF) have
shown inconsistent results. Therefore, understanding the real-time kinetics of gene
expression may be crucial. Here, we use micro positron emission tomography (micro-
PET) to monitor VEGF by imaging the linked PET reporter gene expression.
Methods: 1) Vector: The adenovirus has two expression cassettes with cytomegalovirus
promoters driving VEGF121 and herpes simplex virus type 1 mutant thymidine kinase
reporter gene (Ad-CMV-VEGF121-polyA-CMV-HSV1-sr39tk-polyA). 2) Cell Culture: Cor-
relation of VEGF121 and HSV1-sr39TK was established in transfected rat H9c2 cardi-
omyoblasts using ELISA and [3H]-penciclovir assays, respectively. 3) In Vivo Imaging:
Spraque Dawley rats (n=6) were injected with 1x1010 pfu via thoracotomy. Control rats
(n=6) received Ad-CMV-HSV1-sr39tk, without the linked VEGF121. The same animals
were scanned for >2 weeks using [18F]-FHBG (2.18±0.29 mCi) as PET reporter probe,
followed by a second viral injection at day 30 to assess the efficacy of repeated gene
delivery. Data are expressed as mean±s.e.m.
Results: 1) Transfected H9c2 yield robust VEGF121 and HSV1-sr39TK expressions:
1x1011 pfu (6880721 pg/ml and 5.856 %conversion/ug protein/min), 1010 (491472 and
0.333), 109 (44355 and 0.054), and control (362 and 0.010). Correlation is 0.95. 2) Micro-
PET imaging at day 1 (0.311±0.021 %ID/g), 3 (0.270±0.021), 5 (0.248±0.008), 9
(0.115±0.012), and 15 (0.036±0.008) differs significantly from day 17 (0.028±0.017) and
control (0.025±0.007) (p<0.05). Repeat injections were imaged at day 2 (0.035±0.008)
and 4 (0.026±0.009). Control rats also had similar expression pattern. In vivo results
were confirmed by ex vivo gamma counting, immunohistochemistry, and autoradiogra-
phy.
Conclusion: This is the first cardiac PET study to non-invasively image a linked thera-
peutic and reporter gene construct. Adenoviral mediated gene expression peaked at day
1-3 and lasted only 2 weeks, with no additional expression derived from repeated injec-
tions. Applying this imaging technology to human studies will greatly contribute to the
evaluation of safety and efficacy profiles of cardiac gene therapy.
9:30 a.m.
805-2 Repeated Noninvasive Measurement of pO2 by a Novel 
Electron Paramagnetic Resonance Method in a Rabbit 
Model of Hindlimb Ischemia
Karen L. Moodie, Oleg Y. Grinberg, Huagang Hou, Jane Tomaszewski, Stalina A. 
Grinberg, Eugene Demidenko, Bruce J. Friedman, Harold M. Swartz, Mark J. Post, 
Dartmouth Medical School, Hanover, NH, Maastricht University, Maastricht, The 
Netherlands
Background: Current non-invasive methods to assess blood flow and local oxygen ten-
sion in tissue are limited by high variability, low sensitivity or small penetration depth. This
has been an obstacle to the evaluation of strategies to reverse severe limb ischemia.
Methods: EPR pO2 measurements (using previously injected Char) were obtained pre, 1
day and 14 days after femoral artery ligation. Char is inert but its EPR spectrum changes
in response to oxygen. Regional blood flow (RBF) was measured with microspheres (15
µm BioPal) injected into the left ventricle.
Results: Regional blood flow measurements indicated similar pre and post ligation flows
in the control hind limb, significantly reduced flow in the ligated leg immediately post liga-
tion with normal flow restored by two weeks. EPR oximetry was able to differentiate the
pO2 values in the muscles examined and additionally found significantly reduced pO2 val-
ues pre and post ligation in the treated leg with pO2 returning to just 55% of baseline by
two weeks. Using a mixed model of linear regression analysis, the correlation coefficient
of pO2 and RBF was r=0.42, p<0.01.
Conclusion. EPR oximetry has proven applicable for repeated non-invasive pO2 mea-
surement in the rabbit model of hind limb ischemia. Additionally, results indicate that
blood flow and pO2 show a linear correlation immediately post ischemia. This technique
will be a valuable addition to hindlimb ischemia models and will help in understanding the
physiologic relation between oxygenation and RBF.
9:45 a.m.
805-3 The Modulating Role of CD44 During Arteriogenesis: An 
Experimental and a Clinical Approach
Niels van Royen, Michiel Voskuil, Imo Hoefer, Marco Jost, Ivo Buschmann, Christoph 
Bode, Wolfgang Schaper, Ronald van der Neut, Steven Pals, Jan J. Piek, Academic 
Medical Center, Amsterdam, The Netherlands, University of Freiburg, Freiburg, Germany
Introduction and hypothesis: Recently we have shown that CD44 is strongly expressed
during arteriogenesis in mice and that the absence of this receptor strongly inhibits this
process. In the current study we have further explored the mechanisms through which
CD44 controls arteriogenesis. Perivascular leukocytes as well as FGF-2 and PDGF-B
expression were determined in CD44 null and wildtype mice. In addition, we determined
CD44 expression on activated monocytes in patients with coronary artery disease.
Methods: Using immunohistochemistry, the protein expression of FGF-2 and PDGF-B
was determined in proliferating collateral arteries (day 7 after femoral artery ligation) from
CD44 null and C57BL/6J wildtype mice. mRNA content of the aforementioned growth
factors was then determined with quantitative RT-PCR of laser micro-dissected collateral
arteries. Perivascular leukocytes were identified with CD11b. In patients, the expression
of CD44 was measured on activated monocytes and correlated to the pressure derived
collateral flow index.
Results: In control mice, both FGF-2 and PDGF-B were expressed strongly in the vessel
wall of collateral arteries. The expression of these growth factors was almost abolished in
the CD44 null mice. In contrast, on the mRNA level, no reduction was found (mRNA ratio
of CD44 to wildtype, FGF-2: 3.4, PDGF-B: 2.9). The number of perivascular leukocytes
was significantly reduced in CD44 null mice (wildtype: 29% ± 12% vs. CD44 null: 18% ±
7% CD11b positive cells/square, P<0.01). FACS-analysis showed a significantly lower
CD44 expression on stimulated monocytes from patients with a poor collateralization
(CFI=<0.25) as compared to patients with a good collateralization (CFI>0.25)(poor collat-
eralization: 1764±572 vs. good collateralization: 2817±1029, arbitrary units, P<0.05).
Conclusion: This study shows that CD44 is required for leukocyte extravasation and the
maintenance of FGF-2 and PDGF-B protein expression during collateral artery growth.
Moreover, a strong CD44 expression on stimulated monocytes is related to a well devel-
oped collateral network in patients.
 PR (-) (n=31)  PR (+) (n=35)  P
Sex (M) (%)  65  66  NS
Age  66.2 ± 8.9  66.6 ± 9.8  NS
Stroke (%)  10  14  NS
Myocardial Infarction (%)  19  51  0.007
Hypertension (%)  71  77  NS
Diabetes (%)  36  49  NS
Hyperlipidemia (%)  87  80  NS
Smoking (%)  13  13  NS


805-2 Repeated noninvasive measurement of pO2by a novel electron paramagnetic resonance method in a rabbit model of hindlimb ischemia 

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805-2 Repeated noninvasive measurement of pO2by a novel electron paramagnetic resonance method in a rabbit model of hindlimb ischemia

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