594 Delivery of Recombinant Adeno Associated Virus Mediated Human Tissue Kallikrein Gene Attenuates Insulin Resistance and Prevents Renal Complications in Diabetic Rats Molecular Therapy �������� ���[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
CARDIOVASCULAR
Neovasculature Formation in Ischemic
Myocardium
Karen L Christman,1 Qizhi Fang,2 Richard E Sievers,3 Anne J
Kim,1 Hubert H Fok,3 Albert F Candia,4 Kenneth J Colley,4
Randall J Lee.2,3
1 Joint Bioengineering Graduate Group, University of California
Berkeley and San Francisco, San Francisco, CA, United States;
2 Cardiovascular Research Institute, University of California San
Francisco, San Francisco, CA, United States; 3 Medicine,
University of California San Francisco, San Francisco, CA,
United States; 4 Angiogenix, Inc., Burlingame, CA, United States.
Pleiotrophin (PTN) is a secreted heparin-binding cytokine with
diverse functions In vitro, PTN has been shown to induce
angiogenesis and stimulate endothelial cell proliferation We have
recently demonstrated that the PTN gene is upregulated during
ischemia in the heart, thus indicating a role in angiogenesis in the
myocardium In this study, we hypothesized that delivery of PTN
to ischemic myocardium will induce neovasculature formation We
first developed a pCMV plasmid encoding the PTN gene and
demonstrated via an ELISA that mammalian cells transfected with
this plasmid are capable of producing and secreting PTN The
produced PTN also induced proliferation of serum-starved SW13
cells, indicating that it is biologically active 250 μg of the
pCMV-PTN plasmid was injected directly into the infarcted myocardium
of female Sprague-Dawley rats (n=3) following occlusion of their
left anterior descending portion of the left coronary artery for 17
minutes Another set of rats (n=2) was injected with 250 μg of a
pCMV-β-gal plasmid to serve as a control The rats were sacrificed
after a minimum of five weeks The hearts were rapidly excised,
fresh frozen, and sectioned into 10 μm slices 5 sections from each
heart were stained for capillaries using a Griffonia simplicifolia lectin
which binds to a carbohydrate domain on endothelial cells The
average number of capillaries within the infarct scar was quantified
for each group using Fovea 2.0 plug-in for Adobe Photoshop
Another five sections were stained for arterioles using an anti-smooth
muscle actin antibody The average number and diameter of arterioles
within each infarct were also quantified for each group using SPOT
3.5.5 software Injection of PTN plasmid induced formation of
neovasculature compared to the β-gal plasmid control The capillary
density following delivery of PTN plasmid significantly increased
to 1258±157 capillaries/mm² compared to 782±166 capillaries/mm²
withβ-gal plasmid (P<0.05) The arteriole density also increased to
11±2 arterioles/mm² compared to 4±0 arterioles/mm² for the control
(P<0.03) (see Figure; A: β-gal plasmid, B: PTN plasmid) The
average arteriole diameter was not significantly different In addition,
there was no macroscopic or histological evidence of tumor formation
with the PTN plasmid PTN plasmid may therefore be a possible
treatment for ischemic heart disease
Figure 1 Anti-smooth muscle actin labeled arterioles within the
infarct scar (40×)
Adeno-Associated Virus-Mediated Human Tissue Kallikrein Gene Attenuates Insulin Resistance and Prevents Renal Complications in Diabetic Rats
Gang Yuan,1 Chunxia Zhao,1 Tao Wang,1 Xiao Xiao,1 Juan Li,1 Lee Chao,2 Julie Chao,2 Dao Wen Wang.1
1 Dept of Internal Medicine, Tongji Hospital/Medical College of Huazhong University of Science and Technology, Wuhan, China;
2 Dept of Biochemistry, Medical University of South Carolina, Charleston, SC.
Background: Insulin resistance plays a major role in type 2
diabetes Previous studies have shown that bradykinin potentiates insulin-stimulated glucose uptake and enhances insulin signal through B2 receptor In this study, we evaluated the potential of adeno-associated viral vector (AAV) expressing HK gene as a sole therapy
to correct insulin resistance and to prevent renal complications in
type 2 diabetic rats Methods: Male wistar rats received intravenous
injection of low dose streptozotocin (25mg/kg) and be fed with diets enriched in both glucose and fat to form type 2 diabetic model Single dose of rAAV(1x1010 pfu)expressing HK or LacZ was injected into diabetic rats through tail vein after 4 weeks The caudal arterial pressure was measured using tail cuff device once a week for 16 weeks and 24-hour urine and fasting blood were collected to determine levels of blood glucose, insulin and HK concentrations every 4 weeks until the animals were sacrificed The main organs
were obtained for histological analysis Results: (1) The 4-week
feeding with diets enriched in both glucose and fat induced an increase
in systolic pressure up to 20-25mmHg (95±5.1 up to 124.1±3.9mmHg) and hyperglycemia and hyperinsulinemia All the animals were randomly divided into two groups, receiving rAAV•HK and rAAV•LacZ injection, respectively (2) Delivery of rAAV•HK resulted in a reduction in blood pressure about 20mmHg in 2 weeks and the hypotensive effect lasted for the duration of the study, whereas the blood pressure in control group did not change (Fig 1) (3) The levels of the fast blood insulin were very significantly lower
in rAAV.HK group than control group (HK vs control: 7.72±3.31 mIu/L vs 13.06±4.22 mIu/L,P<0.01) though the levels of fasting blood glucose were not statistically different in both groups (HK vs control: 13.58±3.05 mmol/L vs 13.09±2.14mmol/L) at the end of the study, which suggests that HK enhance insulin sensitivity (4) The expression of HK was detected in blood and urine by ELISA in the HK group but undetectable in control group (5) Histological assessment of kidneys showed near normal morphology in rat kidneys in HK group, but glomerular hypertrophy, mild diffuse mesangial expansion, extracellular matrix accumulation and hyalinosis
in control group These indicated that rAAV•HK gene delivery prevented or reversed the renal injuries caused by diabetes
Conclusions: Our studies demonstrated that the rAAV-mediated
HK gene delivery can very efficiently attenuate insulin resistance and renal injuries as well as lower elevated blood pressure in type 2 diabetes These results suggest that rAAV-mediated HK gene administration may represent a useful tool for the therapy of insulin resistance and the prevention of chronic complications in patients with diabetes
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
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CARDIOVASCULAR
The patent is pending
Improves the Angiogenic Potential of Myoblasts
Louis-Georges Guy,1 Anouk S Fortin,1 Guy Leclerc.1
1 Angiogene Inc., Montreal, QC, Canada.
Myocardial infarction caused by hypoxia results in cardiac tissue
damage Cardiac function can be improved by cell transplantation in
the scarred myocardium Furthermore, the implanted cells can also
be used as vectors for the production of angiogenic factors to improve
perfusion This study examined the angiogenic activity of the Hypoxia
Inducible Factor EPAS1 modified human myoblasts Methods:
Adenoviruses encoding EPAS1 were used to transduce cell lines or
primary myoblasts VEGF induction was used as a marker of
angiogenic activity and was quantified by ELISA and RNase
protection assays Angiogenesis was assayed in vivo by
subcutaneous implantation in mice of Matrigel pellets containing
unmodified or transduced myoblasts Microarrays were used to
document gene induction Results: In the cell types studied, wild
type EPAS1 was found to be a potent VEGF inducer, both in
normoxia and hypoxia In vivo, myoblasts transduced with EPAS1
showed a significantly increased angiogenesis activity in
sub-cutaneous implants Several genes implicated in the different steps
of the angiogenesis process were found to be induced in myoblasts
by EPAS1 Conclusions: EPAS1 is a potent inducer of angiogenic
genes in myoblasts, resulting in a strong in vivo angiogenesis It is
thus a good candidate for genetic manipulation of myoblasts to be
transplanted in the scarred myocardium
Employees of Angiogene Inc
Reendothelialization and Inhibition of In-Stent
Intimal Hyperplasia in Rabbit Model
Yaxue Shi,1 Zhengfeng Wang,1 Hao Zhang,1 Baigen Zhang.1
1 Vascular Surgery, RenJi Hospital of Shanghai Second Medical
University, Shanghai, China.
Objectives: Intact endothelial layer is important to maintain the
smooth muscle cell (SMC) phenotype and prevent the thrombosis
which is related to SMC proliferation.We transfer the
pSV-VEGF165 which stimulates the proliferation of endothelial cells to
the carotid artery by stents and assess its effects on the
reendothelialization and neointima.Methods:28 Cholesterol-fed New
Zealand White rabbits were balloon-denuded and implanted
pSV-VEGF165(800μg) coated stents in the carotid artery.Plasmid
pSV-β-gal was used as control RT-PCR, immunocytochemistry ,electron
microscope, histology were detected Results: After 1d, RT-PCR
showed the expression of VEGF165 mRNA in treatment group(0.3114±0.0493) Its expression increased after 1w (1.0467±0.0670) and persisted till 1m (fig1) Immunocytochemistry also confirmed the expression of VEGF protein, which was found after 3d(20.4950±2.8711%) in media(fig2) and reached the peak after 1w(43.5433±7.8592%) ,attenuated after 3m(9.5200±1.4127%).Scanning electron microscope showed that the time of reendothelialization in treatment group was 1 month (fig3)which was shorter than control group But transmission electron microscope showed that in treatment group there still were synthetic SMCs in media and intima after 1 month ,which was similar to the control group.The intima/media ratio was no difference
between two group(P>0.05)(tab1)(fig4) Conclusions:Plasmid
coated stent can locally deliver the pSV-VEGF165 gene to the stenting site and the vascular cells could uptake the gene,excrete the protein which can promote the endothelialization,but it can not prevent the in-stent intimal hyperplasia
Intimal/media ratio of treatment group and control group control group treatment group
1m 0.50±0.02 0.50±0.04 2m 0.90±0.11 0.87±0.08 3m 1.54±0.34 1.58±0.35
Growth Factor and Prostacyclin Synthase Gene Enhanced Therapeutic Angiogenesis in Rat and Rabbit Ischemia Model
Hiromi Koike,1 Naruya Tomita,2 Tadashi Tanabe,3 Yasufumi Kaneda,1 Ryuichi Morishita.1
1 Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 2 General Medicine, Osaka University Hospital, Suita, Osaka, Japan; 3 Pharmacology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Background: Current therapeutic angiogenesis strategy to treat ischemic disease using angiogenic growth factors has been limited to use a single gene However, as vasodilative substances such as prostacyclin are widely used for the treatment of peripheral arterial disease, we are sure it might be useful to combine the angiogenesis with vasodilation of new vessels Thus, in this study, we examined the angiogenic activity of co-transfection of genes of an angiogenic growth factor, hepatocyte growth factor (HGF), and a vasodilative substance, prostacyclin, synthase
Methods and Results: Ten days after induction of hindlimb ischemia in mice, we intramuscularly injected naked plasmid of human HGF gene and/or prostacyclin synthase gene At 4 weeks after injections of plasmids, co-transfection of HGF gene with prostacyclin synthase gene demonstrated further increase in blood flow as compared to a single gene transfection, accompanied by a significant increase in capillary density, while blood flow was