| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Departments of Medicine (Cardiology) and Biomedical
Research,*
St. Elizabeth's Medical Center of Boston, Tufts
University School of Medicine, Boston, Massachusetts, and the
Department of Protein Therapeutics,
Human
Genome Sciences, Inc., Rockville, Maryland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-nitro-L-arginine methyl
ester. Both VEGFR-2 and VEGFR-3 receptors were shown to be
expressed in human saphenous vein and internal mammary artery. The
potential for VEGF-C to promote angiogenesis in vivo was
then tested in a rabbit ischemic hindlimb model. Ten days after
ligation of the external iliac artery, VEGF-C was administered
as naked plasmid DNA (pcVEGF-C; 500 µg) from the polymer coating of
an angioplasty balloon (n = 8 each) or as
recombinant human protein (rhVEGF-C; 500 µg) by direct intra-arterial
infusion. Physiological and anatomical assessments of angiogenesis 30
days later showed evidence of therapeutic angiogenesis for both
pcVEGF-C and rhVEGF-C. Hindlimb blood pressure ratio (ischemic/normal)
after pcVEGF-C increased to 0.83 ± 0.03 after pcVEGF-C
versus 0.59 ± 0.04 (P <
0.005) in pGSVLacZ controls and to 0.76 ± 0.04 after rhVEGF-C
versus 0.58 ± 0.03 (P < 0.01)
in control rabbits receiving rabbit serum albumin. Doppler-derived
iliac flow reserve was 2.7 ± 0.1 versus 2.0
± 0.2 (P < 0.05) for pcVEGF-C
versus LacZ controls and 2.9 ± 0.3
versus 2.1 ± 0.2 (P < 0.05)
for rhVEGF-C versus albumin controls. Neovascularity was
documented by angiography in vivo (angiographic scores:
0.85 ± 0.05 versus 0.51 ± 0.02
(P < 0.001) for plasmid DNA and 0.74 ± 0.08
versus 0.53 ± 0.03 (P < 0.05)
for protein), and capillary density (per mm2) was
measured at necropsy (252 ± 12 versus 183 ±
10 (P < 0.005) for plasmid DNA and 229 ± 20
versus 164 ± 20 (P < 0.05)
for protein). In contrast to the results of gene targeting
experiments, constitutive expression of VEGF-C in adult animals
promotes angiogenesis in the setting of limb ischemia. VEGF-C and its
receptors thus constitute an apparently redundant pathway for postnatal
angiogenesis and may represent an alternative to VEGF-A for strategies
of therapeutic angiogenesis in patients with limb and/or myocardial
ischemia.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The recent purification and characterization of proteins with high homology to VEGF led to the designation of a vascular endothelial family of growth factors, consisting of four members to date. In addition to VEGF/vascular permeability factor, otherwise renamed VEGF-A or VEGF-1, two additional proteins, VEGF-C or VEGF-220 and VEGF-B or VEGF-3,21 were isolated and shown to be abundantly expressed in highly vascularized tissues such as heart and skeletal muscle. In contrast to the widespread distribution of the VEGFs, the fourth member of the VEGF family, placenta growth factor (PlGF),22 appears restricted in vivo to placenta and certain tumors.23,24 All four proteins share structural homology among themselves as well as with the platelet-derived growth factor A and B polypeptides, including in particular a conserved motif consisting of eight cysteine residues in the putative receptor-binding domain.25 Similarly to the A and B chains of platelet-derived growth factor, VEGF-A and PlGF can form heterodimers with demonstrable biological activity.26-29 VEGF-B may also heterodimerize with VEGF-A in cells expressing both factors.21 Moreover, it was recently shown that hypoxia, a principal stimulus for upregulation of VEGF-A,18,30,31 may differentially regulate the induction of homo- and heterodimers of PlGF/VEGF-A.28
Whereas VEGF-A is a high-affinity ligand for the receptors VEGFR-1 and VEGFR-2, VEGF-C was shown to bind another recently identified endothelium-specific receptor tyrosine kinase, VEGFR-3 (Flt-4); in addition, VEGF-C binds to VEGFR-2, but not to VEGFR-1.20 Comparison of the amino acid sequence of an independently isolated specific activator of VEGFR-3, originally named vascular endothelial growth factor-related protein,32 revealed its identity with VEGF-C. PlGF was shown to bind with high affinity to VEGFR-1, but not to VEGFR-226 or VEGFR-3.33 The receptor(s) for VEGF-B21 has not yet been characterized.
The role of VEGF-A in angiogenesis has been studied extensively in vivo and in vitro.34 More recently, VEGF-A has been shown to be expressed in the media of human arteries and veins,35 consistent with its proposed role in maintaining integrity of the vascular endothelium. The constitutive coexpression of all three VEGF types in many adult tissues20,21,32 suggests an interactive or at least redundant capacity of the VEGF members to regulate angiogenesis and modulate EC function.
Data regarding the bioactivity of VEGF-C and VEGF-B are limited to date.20,21,32,36 Accordingly, we sought to determine whether VEGF-C could promote angiogenesis when overexpressed in adult animals. Experiments in the present study confirm the mitogenic activity of VEGF-C and show its ability to stimulate cell migration for different EC types. VEGF-C is furthermore shown to stimulate release of nitric oxide (NO) from ECs, potentially an important mediator of VEGF-induced angiogenesis.37-40 Indeed, when administered in vivo as plasmid DNA or recombinant protein, VEGF-C was shown to enhance vascular permeability and promote angiogenesis in a rabbit model of hindlimb ischemia. These findings thus indicate that VEGF-C may have utility as a primary or adjunctive agent for therapeutic angiogenesis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Heterodimeric recombinant human (rh) VEGF-A (VEGF-1) protein, purified from Escherichia coli, was a generous gift of Drs. N. Ferrara, B. Keyt, and S. Bunting at Genentech Inc. (South San Francisco, CA). E. coli-derived rhVEGF-A appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 39.8 kd under nonreducing conditions. rhVEGF-C (VEGF-2) was expressed in the BaculoGold Virus Expression Vector System (Pharmingen) and purified by ion exchange and hydrophobic interaction chromatography as described before.41 Under nonreducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded bands of 52 kd and 90 kd, representing multimeric, disulfide-linked rhVEGF-C complexes.20 For all recombinant proteins, endotoxin levels were nondetectable using the limulus lysate assay.
Plasmids
Complementary DNA clones for rhVEGF-C, isolated from a human osteoclast cDNA library, were assembled into the pcDNAI/Amp mammalian expression vector that includes a cytomegalovirus promoter/enhancer to drive gene expression. This plasmid construct was named pcVEGF-C. The plasmid pGSVLacZ (courtesy of Dr. C. Bonnerot), containing a nuclear targeted ß-galactosidase sequence coupled to the SV40 early promoter, was used for control transfection experiments.42
Cell Culture
Human umbilical vein ECs (HUVECs) were isolated from umbilical cord vein by collagenase treatment as previously described43 and grown in medium 199 (Life Technologies, Inc., Grand Island, NY) supplemented with 20% fetal bovine serum (FBS) (Life Technologies), 100 µg/ml EC growth supplement, and 50 U/ml heparin (Sigma Chemical Co., St. Louis, MO). HUVECs were used between passages 1 and 5.
Human microvascular (HM) ECs of dermal origin were purchased from Cascade Biologics, Inc. Immunostaining of representative plates with the antibody to PAL-E44 was used to confirm the absence of lymphatic ECs. The cells were grown in EBM medium containing human epidermal growth factor (10 ng/ml), hydrocortisone (10 ng/ml), 5% FBS, and 0.4% bovine brain extract. HMECs were used between passages 4 and 6.
EC Proliferation Assay
Mitogenic activity was assayed using a previously validated45 colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay with the electron coupling reagent phenazine methosulfate (CellTiter 96 AQ; Promega, Madison, WI). Cells were seeded in a 96-well plate (5000 cells/well) in 0.1 ml serum-supplemented medium and allowed to attach overnight. VEGF (rhVEGF-A or rhVEGF-C) in 5% FBS with heparin (10 U/ml) was added to the wells for 48 hours. MTS/phenazine methosulfate mixture (20 µl; 1:0.05) was added per well and allowed to incubate for 1 hour at 37°C before absorbance was measured at 490 nm in an enzyme-linked immunosorbent assay plate reader. Background absorbance from control wells was subtracted, and seven wells were performed in parallel and averaged for each condition. Proliferation experiments were each repeated three times.
EC Migration Assay
EC migration assays were performed using a 48-well microchemotaxis chamber (Neuroprobe).46 Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 µm (Nuclepore) were coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances were diluted to appropriate concentrations in medium 199 supplemented with 1% FBS, and 25 µl of the final dilution was placed in the lower chamber of a modified Boyden chamber. Subconfluent HUVEC or HMEC cultures were washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chambers, 2.5 x 105 cells suspended in 50 µl medium 199 containing 1% FBS were seeded in the upper compartment. The apparatus was then incubated for 5 hours at 37°C in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter was removed, and the upper side of the filter containing the nonmigrated cells was scraped with a rubber policeman. The filters were fixed with methanol and stained with Giemsa solution (Diff-Quick, Baxter Diagnostics, McGaw Park, IL). Migration was quantified by counting cells in three random high-power fields (x100) in each well. All groups were studied in quadruplicate.
NO Measurement
An NO-specific polarographic electrode connected to an NO meter
(Iso-NO, World Precision Instruments, Sarasota, FL)47
was
used to measure NO released from a cultured HUVEC monolayer.
Calibration of the NO electrode was performed according to the
following equation:
 | (1) |
Miles Assay
Hairless male albino guinea pigs (200 to 600 g; Charles River Breeding Laboratories, Wilmington, MA) were lightly anesthetized with ether (Fisher Scientific, Fair Lawn, NJ). Evans blue dye (Sigma Chemical Co.) was filtered through 0.2-µm micropore filters (Corning Glass, Corning, NY) and then infused as a 0.5- to 1.0-ml bolus of 0.5% solution in saline via the left femoral vein. Evans blue dye binds to circulating plasma proteins and extravasates in response to certain reagents, rendering hyperpermeable dermal sites blue.49 After the animals regained consciousness, reagents were injected intradermally in a volume of 0.1 ml. Intradermal injections were made into the trunk, posterior to the shoulder, 20 minutes after intravenous injection of Evans blue dye with a 30-gauge needle (Becton-Dickinson) causing a bleb of 9 to 11 mm in diameter. Increase in vascular permeability was assessed by leakage of blue dye into the bleb.49 As originally described by Miles and Miles,49 a small area of traumatic bluing 1 to 3 mm in diameter may be seen at the center of the bleb after intradermal injection of saline control. Intensity and area of the blue color changes within the blebs were identified and assessed, and the site of intradermal injection was photographed. The following reagents were injected intradermally: 1) saline alone (as vehicle control), 2) increasing concentrations of rhVEGF-A (4, 8, 16, 32, 64, and 128 ng), and 3) increasing concentrations of rhVEGF-C (4, 8, 16, 32, 64, and 128 ng).
To investigate the role of NO on VEGF-induced vascular permeability,
N-nitro-L-arginine methyl
ester (L-NAME; 20 mg/kg; Sigma Chemical Co.) was
injected via the femoral vein immediately before administration of
Evans blue dye. Twenty minutes later, the following were injected
intradermally: 1) saline alone (negative control), 2) increasing
concentrations of rhVEGF-A (8, 16, 32, 64, and 128 ng), and 3)
increasing concentrations of rhVEGF-C (8, 16, 32, 64, and 128 ng).
Analysis of VEGF Receptor mRNA in Explanted Arterial and Venous Specimens
Total RNA from unused segments of human internal mammary artery and saphenous vein was isolated by phenol/chloroform extraction,50 and RNA concentration was calculated from absorbance at 260 nm.
Reverse transcription51 was performed by heating a 10-µl reaction mixture containing 1 µg total RNA and 20 µg/ml oligodeoxy-thymidine (Life Technologies) at 70°C for 10 minutes. After cooling, 60 units of human placenta ribonuclease inhibitor (Promega) and 200 U Moloney's murine leukemia virus RNase H reverse transcriptase (Life Technologies) were added in a final 20-µl reaction mixture containing (mmol/L) deoxynucleotide triphosphate (Pharmacia) 1 each, dithiothreitol 10, Tris-HCl (pH 8.3) 25, KCl 75, and MgCl2 3; incubated for 1 hour at 42°C; heated 5 minutes at 95°C; and diluted to 50 µl with double-distilled water. For polymerase chain reaction (PCR) with a thermostable DNA polymerase,52 a 50-µl reaction containing 5 µl of cDNA-RNA hybrids, 200 µmol/L deoxynucleotide triphosphate (Pharmacia), 20 mmol/L Tris-HCl (pH 8.55), 2.5 mmol/L MgCl2, 16 mmol/L (NH4)2SO4, 150 µg/ml bovine serum albumin, 1 µmol/L each oligonucleotide primer, and 1.25 U of Taq polymerase (Perkin-Elmer Corp., Norwalk, CT) was subjected to the following temperature cycles: 30 seconds of denaturing at 95°C, 5 seconds of annealing at 56°C, and 1 minute of extension at 72°C. At the end of the last cycle, a prolonged extension step was carried out for 10 minutes. PCR products were analyzed by electrophoresing 10 µl of each PCR reaction mixture in a 1.5% agarose gel, and bands were visualized by ethidium bromide staining. The results represent three independent amplifications from two separate studies.
The primers chosen for human VEGFR-3 (GenBank accession number X68203) were: for sense (5' to 3'), CAG GAT GAA GAC ATT TGA, and for antisense (5' to 3'), AAG AAA ATG CTG ACG TAT GC53 (190 bp PCR-product); for human VEGFR-2 (GenBank accession number X61656), CAA CAA AGT CGG GAG AGG AG and ATG ACG ATG GAC AAG TAG CC (819 bp); for human VEGFR-1 (GenBank accession number X51602), CAG CGG CTT TTG TGG AAG ACT CAC and ACA TCT CGG TGT CAC TTC TTG GAC (735 bp); and for human glyceraldehyde-3-phosphate dehydrogenase, TGA AGG TCG GAG TCA ACG GAT TTG and CAT GTG GGC CAT GAG GTC CAC CAC (983 bp). The number of cycles was 40 for VEGFR-1, VEGFR-2, and VEGFR-3, and 30 for glyceraldehyde-3-phosphate dehydrogenase.
Animal Model
The potential for VEGF-C to promote angiogenesis in vivo was investigated in a previously described rabbit model of hindlimb ischemia.54,55 All protocols were approved by the Institutional Animal Care and Use Committee of St. Elizabeth's Medical Center. A total of 28 (seven per group) male New Zealand White rabbits (3.5 to 4.0 kg) (Pine Acre Rabbitry) were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8 mg/kg) after premedication with xylazine (2 mg/kg). The femoral artery of one hindlimb was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates into the saphenous and popliteal arteries. Consequently, blood flow to the ischemic limb is dependent on collateral vessels originating from the internal iliac artery.54,55 An interval of 10 days was allowed for postoperative recovery and development of endogenous collateral vessels. Beyond this time point, studies performed up to 90 days postoperatively55 have demonstrated no significant collateral vessel augmentation.
Percutaneous Arterial Gene Transfer of pcVEGF-C
At 10 days postoperatively (day 0), after performing a baseline angiogram (see below), the internal iliac artery of the ischemic limb of eight animals was transfected with pcVEGF-C as naked DNA using a 2.0-mm hydrogel-coated balloon catheter (Slider with Hydroplus, Boston Scientific, Natick, MA). The angioplasty balloon was prepared ex vivo by first advancing the deflated balloon through a 5-F Teflon sheath (Boston Scientific), applying 500 µg of pcVEGF-C solution to the 20 µm-thick layer of hydrogel coating the external surface of the inflated balloon, and then retracting the inflated balloon back into the protective sheath. The sheath and angioplasty catheter were then introduced via the right carotid artery and advanced to the lower abdominal aorta using a 0.014-inch guide wire (Hi-Torque Floppy II, Advanced Cardiovascular Systems) under fluoroscopic guidance. The balloon catheter was then advanced into the internal iliac artery of the ischemic limb, inflated for 1 minute at 6 atmospheres, deflated, and withdrawn. An identical protocol was used to transfect the internal iliac artery of control animals (n = 8) with the promoter-matched plasmid pGSVLacZ containing a nuclear targeted ß-galactosidase sequence.
On day 30, all measurements were repeated (see below), the animals were sacrificed, and tissue samples were retrieved for histological examination.
Intra-arterial Administration of rhVEGF-C Protein
Ten days postoperatively (day 0), baseline noninvasive and invasive hemodynamic parameters (see below) were recorded. A single intra-arterial bolus of rhVEGF-C (500 µg; n = 8) in 3 ml of phosphate-buffered saline or vehicle solution (3 ml of phosphate-buffered saline with 0.1% rabbit serum albumin (RSA), n = 8) was then administered over a period of 1 minute through a 3-F end-hole infusion catheter (Tracker-18; Target Therapeutics) positioned in the internal iliac artery of the ischemic limb. The catheter was then washed with 3 ml of phosphate-buffered saline containing 0.1% albumin. The dose of rhVEGF-C protein used in the present experiment (500 µg) has previously been shown for rhVEGF-1 to induce a maximal effect using a single bolus protocol.12,56
Physiological Assessment
Calf Blood Pressure Ratio
Calf blood pressure was measured from the posterior tibial artery at day 0 and 30 in both hindlimbs using a Doppler Flowmeter (model 1050, Parks Medical Electronics) and cuff connected to a pressure manometer.54,57 The calf blood pressure ratio was defined for each rabbit as the ratio of systolic pressure of the ischemic limb to that of the normal limb.
Intravascular Blood Flow Measurement
Blood flow was quantified in vivo before selective internal iliac angiography on day 30 with a 0.018-inch Doppler guide wire (Cardiometrics) introduced into the left common carotid artery as previously described.58 The wire was advanced via a 3-F infusion catheter (Target Therapeutics) positioned at the origin of the common iliac artery to the proximal segment of the internal iliac artery supplying the ischemic limb. Average peak velocity (APV) was recorded at rest, and the maximum APV was evaluated after intra-arterial administration of nitroprusside (Sigma Chemical Co.) over 2 minutes via a constant infusion pump (1 ml/minute). The drug was administered at a dose of 1.5 µg/minutes/kg.
Angiographic luminal diameter of the internal iliac artery was determined, at rest and after drug infusion, using an automated edge-detection system (Quantum 2000I; QCS) as previously described.54 The luminal diameter was measured at the site of the Doppler sample volume 5 mm distal to the wire tip. Cross-sectional area was calculated assuming a circular lumen.
Doppler-derived flow was calculated as QD =
(
d2/4)(0.5 x APV), where
QD is Doppler-derived time-averaged flow,
d is vessel diameter, and APV is time average of the
spectral peak velocity.59
The mean velocity was estimated
as 0.5 x APV by assuming a time-averaged parabolic velocity
profile across the vessel. The Doppler-derived flow calculated in this
fashion has been previously validated in vivo.59
For the ischemic limb, in which the external iliac artery had been
removed, flow through the internal iliac artery represented flow to the
entire hindlimb.54
Blood flow reserve was defined as the
ratio of resting to maximum blood flow.
Anatomical Assessment
Selective Iliac Angiography
Angiographic analysis of collateral vessel development in the ischemic limb was performed using the 4-second angiograms recorded after injection of 0.25 mg of nitroglycerin and 5 ml of nonionic contrast medium (Isovue-370; Squibb Diagnostics, New Brunswick, NJ) into the internal iliac artery through a 3-F infusion catheter (Target Therapeutics). A grid overlay comprising 2.5 mm-diameter circles arranged in rows spaced 5 mm apart was placed over the angiogram at the level of the medial thigh. The number of contrast-opacified arteries crossing over circles and the total number of circles encompassing the medial thigh area were counted in single blinded fashion. An angiographic score was calculated as the ratio of circles crossed by opacified arteries divided by the total number of circles in the ischemic thigh. This angiographic score reflects vascular density in the medial thigh.57
Capillary Density
Collateral vessel formation was further examined by measuring the number of capillaries in light microscopic sections taken from ischemic and nonischemic hindlimbs as previously described.57 Tissue specimens were retrieved from each limb as transverse sections from the adductor and semimembranous muscles at the time of sacrifice (day 30). These two muscles were chosen because they are the two major muscles of the medial thigh, and each was originally perfused by the deep femoral artery, ligated at the time of the surgery. Samples were embedded in ornithine carbamoyltransferase compound (Miles Inc., Elkhart, IN) and snap-frozen in liquid nitrogen. Frozen sections (5 µm in thickness) were stained with alkaline phosphatase using an indoxyl-tetrazolium method to detect capillary ECs followed by eosin counterstaining.60 Capillaries were counted under a x20 objective to determine the capillary density (mean number of capillaries/mm2). A total of 20 different fields from the two muscles were randomly selected, and the number of capillaries was counted. To ensure that analysis of capillary density was not overestimated because of muscle atrophy, capillary density was also evaluated as a function of the number of muscle fibers in the histological section (capillary/myocyte ratio).
Statistical Analysis
Results were expressed as mean ± standard error of the mean (SEM). Statistical significance was evaluated using unpaired Student's t-test for comparisons between two means and analysis of variance followed by Scheffé's procedure for more than two means. A value of P < 0.05 was interpreted to denote statistical significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The effects of rhVEGF-C on EC proliferation are shown in Figure 1
. At a concentration of 10 ng/ml, the
mitogenic activity of VEGF-C protein on HUVECs was 35% of that seen
with VEGF-A. This difference was statistically significant
(P < 0.005) (Figure 1A)
. The mitogenic effect
of VEGF-C was more pronounced when applied to HMECs (Figure 1B)
: at 10
ng/ml, the mitogenic response of VEGF-C was 72% as potent as VEGF-A
(P < 0.01).
|
Both VEGF-A and VEGF-C protein exhibited a pronounced chemotactic
effect on ECs. The dose-response curves showed a typical Gaussian
distribution, beginning with an effect on migration that was evident at
a minimal concentration of 0.1 ng/ml (Figure 2)
. For HUVECs, at a concentration of 10
ng/ml, the chemotactic response of VEGF-C was 83% of that seen with
VEGF-A (P < 0.05) (Figure 2A)
; for HMECs, the
chemotactic response to VEGF-A and VEGF-C was indistinguishable (Figure 2B)
.
|
Treatment of HUVECs with VEGF-C (1, 10, and 100 ng/ml) resulted in
a concentration-dependent increase in NO synthesis (Figure 3)
. Peak NO synthesis was observed
between 3 and 6 minutes after addition of VEGF-C. Treatment of HUVECs
with VEGF-A (100 ng/ml) under the same experimental conditions induced
NO synthesis as previously reported.61
S-Nitroso-N-acetyl-penicillamine was
used as a positive control, and 0.1 mmol/L resulted in a significant
increase in NO concentration measured in the buffer medium. For
comparison, peak NO measured in response to VEGF-C was 92.7% and
80.9% of that measured after the same dose of VEGF-A and 0.1 mmol/L
S-nitroso-N-acetyl-penicillamine, respectively.
|
Repeated intradermal injection of the vehicle control saline (0.1
ml) did not increase vascular permeability. Intradermal injection of
either VEGF-A or VEGF-C (16, 32, 64, or 128 ng) significantly increased
vascular permeability in a dose-dependent manner (Figure 4A)
. Development of a positive (blue)
response began 155 ± 6 seconds (n = 3)
after intradermal injection. Repeated experiments
(n = 3) did not show significant differences in
the induction of vascular permeability between VEGF-A and VEGF-C. As a
positive control, we used bradykinine and histamine (100 nmol/L), both
of which increased vascular permeability at the injected sites (data
not shown).
|
. The
inactive stereoisomer D-NAME (20 mg/kg), which does not
inhibit endothelial NO synthesis, failed to inhibit the increase in
vascular permeability observed with either VEGF-1 or VEGF-C (not
shown). Thus, for both VEGF-A and VEGF-C, increased vascular
permeability was dependent on local production of NO. VEGF-C Receptor Expression
To identify potential target vessels for VEGF-C activity in
adults, we studied VEGFR-3 and VEGFR-2 mRNA expression in explanted
segments of human internal mammary artery and saphenous vein by reverse
transcription-PCR. For these experiments we used human specimens,
because the corresponding nucleotide sequences for rabbit species have
not yet been published. Figure 5
demonstrates that the VEGF-C receptors VEGFR-2 and VEGFR-3, as well as
VEGFR-1, are expressed in both venous and arterial tissues. As a
positive control, we used HUVECs, for which coexpression of VEGFR-1,
VEGFR-2, and VEGFR-3 has been previously described.53
RNA
from human fibroblasts, from which amplification never resulted in
positive bands for these receptors, was used as a negative control.
|
Calf blood pressure ratio was similar in all groups at day 0
(Figure 6A)
. By day 30, blood pressure
ratio in the pcVEGF-C and VEGF-C protein-treated groups had increased
to 0.83 ± 0.03 and 0.76 ± 0.04, respectively. In both
cases, this value exceeded that measured for the control groups
(0.59 ± 0.04 and 0.58 ± 0.03; P = 0.002 and
0.009, respectively). Blood pressure ratio in rabbits undergoing gene
transfer with phVEGF-C was not significantly different from that
recorded in rabbits treated with VEGF-C recombinant protein.
|
Iliac flow reserve, the ratio between blood flow at rest and after
maximal pharmacological stimulation, was 2.90 ± 0.31 and
2.65 ± 0.14 for the pcVEGF-C- and rhVEGF-C-treated rabbits,
respectively. In both cases, this represented a statistically
significant increase compared with controls (pGSVLacZ = 2.10
± 0.17 and RSA = 2.04 ± 0.23; P = 0.04 and
P = 0.04, respectively) (Figure 6C)
.
Effect of pcVEGF-C and rhVEGF-C on Anatomical Evidence of Collateral Development
Quantitative analysis of collateral vessel development in the
medial thigh is summarized in Figure 6B
. Before treatment (day 0),
there were no significant differences among groups in angiographic
score. By day 30, the angiographic score in both VEGF-C gene
transfer (0.85 ± 0.05) and protein (0.74 ± 0.08) groups
exceeded that measured in the control groups (pGSVLacZ = 0.51
± 0.02 (P = 0.00003) and RSA = 0.53
± 0.03 (P = 0.023), respectively). The
angiographic score in the pcVEGF-C gene transfer group was
significantly higher than that measured for the protein-treated group
(P = 0.02). Figure 7
shows representative internal iliac
angiograms recorded at day 30 from controls, VEGF-C plasmid, and VEGF-C
recombinant protein-treated animals. In both treatment groups,
collateral artery development was more marked compared with
corresponding control groups.
|
. Analysis of capillary/muscle fiber
ratio yielded similar results (data not shown). Analysis of capillary
density and capillary/muscle fiber ratio in the medial thigh muscle of
the nonischemic limb showed no differences among groups (data not
shown). Representative examples of histological sections stained for
alkaline phosphatase in the different experimental groups are shown in
Figure 8
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The divergence in bioactivity, as shown for VEGF-A and PlGF,26,28 is most likely due to different affinities of the four VEGF members to the three known endothelial-specific fms-like tyrosine kinases VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-C was isolated as a ligand and specific activator of VEGFR-320,32 and, in addition, was shown to stimulate autophosphorylation of VEGFR-2.20 Moreover, VEGFR-3 and VEGFR-2 mRNA expression was demonstrated in cultured HUVECs,53 HMECs, and femoral vein ECs.63 We therefore initiated our evaluation of VEGF-C bioactivity by performing proliferation and migration assays on different EC types in vitro. We observed clear mitogenic and chemotactic responses of ECs to VEGF-C, in agreement with previous reports.20,32 Although the impact on cell migration was similar for VEGF-A and VEGF-C, the proliferative response to VEGF-C was less than that observed for VEGF-A, particularly in the case of HUVECs. Despite the latter, in vivo angiogenesis observed in response to VEGF-C was robust and indistinguishable from that observed previously in the same animal model using VEGF-A recombinant protein12 or plasmid DNA.64
The fact that the in vivo results of these experiments better parallel in vitro analyses of migration as opposed to proliferation is consistent with notions regarding the relative contribution of these activities to angiogenesis. In a classic experiment performed in the rat cornea, Sholley et al65 showed that vascular sprouting could be induced and continue for more than 2 days, despite irradiation treatment sufficient to suppress DNA synthesis. Angiogenic activity in this model was thus interpreted to reflect the dominant impact of EC migration. Alternatively, it is possible that the somewhat one-dimensional in vitro assays commonly used to evaluate EC proliferation and migration do not accurately reflect the potential for angiogenesis to develop in vivo.
NO is released from ECs and arterial segments in cell and organ culture, respectively,58,61 and has been shown to mediate VEGF-induced neovascularization in a rabbit cornea pocket assay.66 More recently, experiments that we have performed in mice lacking endothelial NO synthase67 have established that endogenous NO sulfate is essential for endogenous angiogenesis that develops in response to limb ischemia, and that the absence of endogenous NO sulfate precludes a favorable response to rhVEGF165.67 Our finding that VEGF-C promotes NO release in vitro that is comparable to VEGF-A is consistent with the demonstration that VEGF-C promotes angiogenesis in vivo.
In accordance with this finding, VEGF-C enhanced permeability in the Miles assay, an effect that was almost completely inhibited by prior administration of an inhibitor of NO synthase. We have recently reported similar results for VEGF-A.68 Given the extent to which angiogenesis and permeability appear to be linked,69 this finding provides additional evidence for involvement of NO in mediating VEGF (A and C)-mediated angiogenesis. To our knowledge, the results obtained using the Miles assay establish VEGF-C as the only growth factor other than VEGF-A to augment vascular permeability.
Subsequent to initial descriptions of VEGF-C and VEGF-B20,62 serious questions have been raised regarding the bioactivity of these two gene products. In vitro, both factors were found to exhibit mitogenic activity,20,21,32 and VEGF-C stimulated bovine EC outgrowth in a collagen gel assay.20 Overexpression of VEGF-C in the skin of transgenic mice, however, was observed by Jeltsch et al36 to result in lymphatic vessel hyperplasia; because no evidence of angiogenesis was observed, these findings were interpreted to indicate that VEGF-C does not promote angiogenesis but is instead a specific ligand for lymphatic endothelium.36 As the authors acknowledged, this outcome was "unexpectedly specific," because VEGF-C has been shown to transduce the VEGFR-2 receptor, the principal receptor responsible for VEGF-A-induced angiogenesis.
Our findings contradict the assertion of Jeltsch et al.36 Using a previously established rabbit ischemic hindlimb model,54,55 we demonstrated that VEGF-C clearly promotes angiogenesis in vivo. Anatomical and physiological evidence of augmented angiogenesis observed in this animal model have been previously shown to result from marked enhancement of EC proliferative activity in response to an endothelial mitogen70 and thus implies the development of vascular sprouts. Preclinical studies in this animal model using naked DNA encoding for VEGF-A have been duplicated in human subjects.15,71 Similar to results reported previously for VEGF-A,12,64 VEGF-C—administered as naked DNA or recombinant protein—increased flow reserve, hindlimb perfusion pressure, angiographically visible collateral vessels, and capillary density. In those experiments in which VEGF-A was transferred as plasmid DNA, reverse transcription-PCR followed by Southern blot analysis detected expression of the transferred gene up to 21 days posttransfection in the arterial wall.72 Such continuous expression of the introduced gene constitutes one potential explanation for the more pronounced effect on physiological and anatomical parameters after gene transfer versus single bolus administration of recombinant VEGF-C protein. Alternatively, VEGF-C, once transcribed and translated, may form intracellular heterodimers with other VEGF family members, as shown for VEGF-A27-29 and VEGF-C,21 thereby compounding effects attributable to one factor alone.
There are at least two potential explanations for the differing outcomes observed previously in transgenic mice and those observed currently in the rabbit model. First, physiological function of any ligand is dependent on the temporal and spatial expression of its specific receptors. It remains to be determined which of the two receptors, VEGFR-2 or VEGFR-3, or both, transduce the effects of VEGF-C, given that autophosphorylation of both receptors has been shown to occur after stimulation with VEGF-C,20,32 and both are shown here to be expressed postnatally in segments of arteries and veins. (Similar results have been previously reported in cultured HMECs and femoral and umbilical vein ECs.53,63 ) Even if VEGFR-3 function in adults is limited to maintenance of lymphatic vessels, as demonstrated recently for embryonic development,36,73 postnatal angiogenesis may be exclusively mediated by the VEGFR-2 receptor.36 In addition, the possible formation of VEGFR-2/VEGFR-3 heterodimers could lead to more cell type and/or tissue-specific consequences.36
Furthermore, previous studies from our laboratory74 and those of others75 have shown that ischemia leads to regional upregulation of VEGFR-2, the principal receptor mediating VEGF-A-induced angiogenesis. In the absence of such ischemia-induced upregulation, we have never observed angiogenesis in response to transient overexpression of VEGF-A.12,76 Consequently, in the absence of appropriate receptor upregulation, even transient overexpression of VEGF might have been interpreted not to promote angiogenesis in postnatal animal models.
A second potential explanation involves the construct used by Jeltsch et al36 to generate VEGF-C transgenic mice. Because it utilizes the human keratin 14 (K14) promoter, gene expression is targeted to the basal cells of stratified squamous epithelium. It may thus not be valid to extrapolate these results to gene expression in other tissues or organs.
In summary, the current study demonstrates that despite evidence of specificity for lymphatic endothelium in a developmental model, VEGF-C, when administered to adult animals in the setting of tissue ischemia, promotes angiogenesis and augments flow to ischemic tissues. These findings underscore the principle that constitutive overexpression of a given protein during embryogenesis is not necessarily equivalent to transient overexpression of the same protein postnatally; postnatal receptor modulation associated with specific pathological processes, in this case tissue ischemia, may in part explain the differing outcomes observed in the embryonic versus adult organism. Thus, VEGF-C may indeed have utility as a primary or adjunctive agent for therapeutic angiogenesis.
|
Footnotes |
|---|
Accepted for publication April 24, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  P. Liu, W. Chen, H. Zhu, B. Liu, S. Song, W. Shen, F. Wang, S. Tucker, B. Zhong, and D. Wang Expression of VEGF-C Correlates with a Poor Prognosis Based on Analysis of Prognostic Factors in 73 Patients with Esophageal Squamous Cell Carcinomas Jpn. J. Clin. Oncol., October 1, 2009; 39(10): 644 - 650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Howell, C. M. Costello, M. Sands, I. Dooley, and P. McLoughlin L-Arginine promotes angiogenesis in the chronically hypoxic lung: a novel mechanism ameliorating pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L1042 - L1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kodama, Y. Kitadai, M. Tanaka, T. Kuwai, S. Tanaka, N. Oue, W. Yasui, and K. Chayama Vascular Endothelial Growth Factor C Stimulates Progression of Human Gastric Cancer via Both Autocrine and Paracrine Mechanisms Clin. Cancer Res., November 15, 2008; 14(22): 7205 - 7214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Benest, S. J. Harper, S. Y. Herttuala, K. Alitalo, and D. O. Bates VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis Cardiovasc Res, May 1, 2008; 78(2): 315 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Horvath and Y. Zhou Transmyocardial Laser Revascularization and Extravascular Angiogenetic Techniques to Increase Myocardial Blood Flow Card. Surg. Adult, January 1, 2008; 3(2008): 733 - 752. [Full Text]
|
|
|
|
 |
|
 S. Keskitalo, T. Tammela, J. Lyytikka, T. Karpanen, M. Jeltsch, J. Markkanen, S. Yla-Herttuala, and K. Alitalo Enhanced Capillary Formation Stimulated by a Chimeric Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor-C Silk Domain Fusion Protein Circ. Res., May 25, 2007; 100(10): 1460 - 1467. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tammela, Y. He, J. Lyytikka, M. Jeltsch, J. Markkanen, K. Pajusola, S. Yla-Herttuala, and K. Alitalo Distinct Architecture of Lymphatic Vessels Induced by Chimeric Vascular Endothelial Growth Factor-C/Vascular Endothelial Growth Factor Heparin-Binding Domain Fusion Proteins Circ. Res., May 25, 2007; 100(10): 1468 - 1475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhao, G. Smith, J. Cai, A. Ma, and M. Boulton Vascular endothelial growth factor C promotes survival of retinal vascular endothelial cells via vascular endothelial growth factor receptor-2 Br J Ophthalmol, April 1, 2007; 91(4): 538 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Saito, H. Nakagami, R. Morishita, Y. Takami, Y. Kikuchi, H. Hayashi, T. Nishikawa, K. Tamai, N. Azuma, T. Sasajima, et al. Transfection of Human Hepatocyte Growth Factor Gene Ameliorates Secondary Lymphedema via Promotion of Lymphangiogenesis Circulation, September 12, 2006; 114(11): 1177 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Saaristo, T. Tammela, A. Farkkila, M. Karkkainen, E. Suominen, S. Yla-Herttuala, and K. Alitalo Vascular Endothelial Growth Factor-C Accelerates Diabetic Wound Healing Am. J. Pathol., September 1, 2006; 169(3): 1080 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hoshida, N. Isaka, J. Hagendoorn, E. di Tomaso, Y.-L. Chen, B. Pytowski, D. Fukumura, T. P. Padera, and R. K. Jain Imaging Steps of Lymphatic Metastasis Reveals That Vascular Endothelial Growth Factor-C Increases Metastasis by Increasing Delivery of Cancer Cells to Lymph Nodes: Therapeutic Implications Cancer Res., August 15, 2006; 66(16): 8065 - 8075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B Zhao, A Ma, J Cai, and M Boulton VEGF-A regulates the expression of VEGF-C in human retinal pigment epithelial cells Br J Ophthalmol, August 1, 2006; 90(8): 1052 - 1059. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Karpanen, C. A. Heckman, S. Keskitalo, M. Jeltsch, H. Ollila, G. Neufeld, L. Tamagnone, and K. Alitalo Functional interaction of VEGF-C and VEGF-D with neuropilin receptors FASEB J, July 1, 2006; 20(9): 1462 - 1472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Miyata, S. Kanda, K. Ohba, K. Nomata, Y. Hayashida, J. Eguchi, T. Hayashi, and H. Kanetake Lymphangiogenesis and Angiogenesis in Bladder Cancer: Prognostic Implications and Regulation by Vascular Endothelial Growth Factors-A, -C, and -D Clin. Cancer Res., February 1, 2006; 12(3): 800 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ohtsuka, K. Inoue, Y. Hara, N. Morioka, K. Ohshima, J. Suzuki, A. Ogimoto, Y. Shigematsu, and J. Higaki Serum markers of angiogenesis and myocardial ultrasonic tissue characterization in patients with dilated cardiomyopathy Eur J Heart Fail, June 1, 2005; 7(4): 689 - 695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Madeddu Therapeutic angiogenesis and vasculogenesis for tissue regeneration Exp Physiol, May 1, 2005; 90(3): 315 - 326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Baumgartner, H. C. Thoeny, O. Kummer, C. Roefke, C. Skjelsvik, C. Boesch, and R. Kreis Leg Ischemia: Assessment with MR Angiography and Spectroscopy Radiology, March 1, 2005; 234(3): 833 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Skurk, H. Maatz, E. Rocnik, A. Bialik, T. Force, and K. Walsh Glycogen-Synthase Kinase3{beta}/{beta}-Catenin Axis Promotes Angiogenesis Through Activation of Vascular Endothelial Growth Factor Signaling in Endothelial Cells Circ. Res., February 18, 2005; 96(3): 308 - 318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kurahara, S. Takao, K. Maemura, H. Shinchi, S. Natsugoe, and T. Aikou Impact of Vascular Endothelial Growth Factor-C and -D Expression in Human Pancreatic Cancer: Its Relationship to Lymph Node Metastasis Clin. Cancer Res., December 15, 2004; 10(24): 8413 - 8420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers, A. T. Van Oosterom, and E. A. De Bruijn Vascular Endothelial Growth Factor and Angiogenesis Pharmacol. Rev., December 1, 2004; 56(4): 549 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Uchiba, K. Okajima, Y. Oike, Y. Ito, K. Fukudome, H. Isobe, and T. Suda Activated Protein C Induces Endothelial Cell Proliferation by Mitogen-Activated Protein Kinase Activation In Vitro and Angiogenesis In Vivo Circ. Res., July 9, 2004; 95(1): 34 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Walter, M. Cejna, L. Diaz-Sandoval, S. Willis, L. Kirkwood, P. W. Stratford, A. B. Tietz, R. Kirchmair, M. Silver, C. Curry, et al. Local Gene Transfer of phVEGF-2 Plasmid by Gene-Eluting Stents: An Alternative Strategy for Inhibition of Restenosis Circulation, July 6, 2004; 110(1): 36 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Seipelt, C. L. Backer, C. Mavroudis, V. Stellmach, I. M. Seipelt, M. Cornwell, J. Hernandez, and S. E. Crawford Topical VEGF Enhances Healing of Thoracic Aortic Anastomosis for Coarctation in a Rabbit Model Circulation, September 9, 2003; 108(90101): II-150 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.C Felmeden, A.D Blann, and G.Y.H Lip Angiogenesis: basic pathophysiology and implications for disease Eur. Heart J., April 1, 2003; 24(7): 586 - 603. [Full Text] [PDF]
|
|
|
|
|
|
J. Krishnan, V. Kirkin, A. Steffen, M. Hegen, D. Weih, S. Tomarev, J. Wilting, and J. P. Sleeman Differential in Vivo and in Vitro Expression of Vascular Endothelial Growth Factor (VEGF)-C and VEGF-D in Tumors and Its Relationship to Lymphatic Metastasis in Immunocompetent Rats Cancer Res., February 1, 2003; 63(3): 713 - 722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dodge-Khatami, H.W.M. Niessen, A. Baidoshvili, T.M. van Gulik, M.G. Klein, L. Eijsman, and B.A.J.M. de Mol Topical vascular endothelial growth factor in rabbit tracheal surgery: comparative effect on healing using various reconstruction materials and intraluminal stents Eur. J. Cardiothorac. Surg., January 1, 2003; 23(1): 6 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Losordo and A. Kawamoto Biological Revascularization and the Interventional Molecular Cardiologist: Bypass for the Next Generation Circulation, December 10, 2002; 106(24): 3002 - 3005. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Saaristo, T. Veikkola, T. Tammela, B. Enholm, M. J. Karkkainen, K. Pajusola, H. Bueler, S. Yla-Herttuala, and K. Alitalo Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects J. Exp. Med., September 16, 2002; 196(6): 719 - 730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Smith Jr, K.-F. Lin, J. Agata, L. Chao, and J. Chao Human Endothelial Nitric Oxide Synthase Gene Delivery Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1279 - 1285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. SAARISTO, T. VEIKKOLA, B. ENHOLM, M. HYTONEN, J. AROLA, K. PAJUSOLA, P. TURUNEN, M. JELTSCH, M. J. KARKKAINEN, D. KERJASCHKI, et al. Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes FASEB J, July 1, 2002; 16(9): 1041 - 1049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Jussila and K. Alitalo Vascular Growth Factors and Lymphangiogenesis Physiol Rev, July 1, 2002; 82(3): 673 - 700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. He, K.-i. Kozaki, T. Karpanen, K. Koshikawa, S. Yla-Herttuala, T. Takahashi, and K. Alitalo Suppression of Tumor Lymphangiogenesis and Lymph Node Metastasis by Blocking Vascular Endothelial Growth Factor Receptor 3 Signaling J Natl Cancer Inst, June 5, 2002; 94(11): 819 - 825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Witmer, J. Dai, H. A. Weich, G. F.J.M. Vrensen, and R. O. Schlingemann Expression of Vascular Endothelial Growth Factor Receptors 1, 2, and 3 in Quiescent Endothelia J. Histochem. Cytochem., June 1, 2002; 50(6): 767 - 778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Byzova, C. K. Goldman, J. Jankau, J. Chen, G. Cabrera, M. G. Achen, S. A. Stacker, K. A. Carnevale, M. Siemionow, S. R. Deitcher, et al. Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo Blood, May 29, 2002; 99(12): 4434 - 4442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. K. Ozaki, K. D. Beharry, K. C. Nishihara, Y. Akmal, J. G. Ang, R. Sheikh, and H. D. Modanlou Regulation of Retinal Vascular Endothelial Growth Factor and Receptors in Rabbits Exposed to Hyperoxia Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1546 - 1557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. MAKINEN and K. ALITALO Molecular Mechanisms of Lymphangiogenesis Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 189 - 196. [Abstract] [PDF]
|
|
|
|
 |
|
 G. S. Marchand, N. Noiseux, J.-F. Tanguay, and M. G. Sirois Blockade of in vivo VEGF-mediated angiogenesis by antisense gene therapy: role of Flk-1 and Flt-1 receptors Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H194 - H204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-C. Tille, J. Wood, S.J. Mandriota, C. Schnell, S. Ferrari, J. Mestan, Z. Zhu, L. Witte, and M. S. Pepper Vascular Endothelial Growth Factor (VEGF) Receptor-2 Antagonists Inhibit VEGF- and Basic Fibroblast Growth Factor-Induced Angiogenesis in Vivo and in Vitro J. Pharmacol. Exp. Ther., December 1, 2001; 299(3): 1073 - 1085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Karpanen and K. Alitalo Lymphatic Vessels as Targets of Tumor Therapy? J. Exp. Med., September 17, 2001; 194(6): F37 - F42. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dodge-Khatami, C. L. Backer, L. D. Holinger, C. Mavroudis, K. E. Cook, and S. E. Crawford Healing of a free tracheal autograft is enhanced by topical vascular endothelial growth factor in an experimental rabbit model J. Thorac. Cardiovasc. Surg., September 1, 2001; 122(3): 554 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Skobe, L. M. Hamberg, T. Hawighorst, M. Schirner, G. L. Wolf, K. Alitalo, and M. Detmar Concurrent Induction of Lymphangiogenesis, Angiogenesis, and Macrophage Recruitment by Vascular Endothelial Growth Factor-C in Melanoma Am. J. Pathol., September 1, 2001; 159(3): 893 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Isner, P. R. Vale, J. F. Symes, and D. W. Losordo Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subjects Circ. Res., August 31, 2001; 89(5): 389 - 400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lu, J. Shansky, M. Del Tatto, P. Ferland, X. Wang, and H. Vandenburgh Recombinant Vascular Endothelial Growth Factor Secreted From Tissue-Engineered Bioartificial Muscles Promotes Localized Angiogenesis Circulation, July 31, 2001; 104(5): 594 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Losordo and J. M. Isner Vascular endothelial growth factor-induced angiogenesis: crouching tiger or hidden dragon? J. Am. Coll. Cardiol., June 15, 2001; 37(8): 2131 - 2135. [Full Text] [PDF]
|
|
|
|
|
|
R Tabibiazar and S.G Rockson Angiogenesis and the ischaemic heart Eur. Heart J., June 1, 2001; 22(11): 903 - 918. [PDF]
|
|
|
|
|
|
R. Clarijs, L. Schalkwijk, D. J. Ruiter, and R. M. W. de Waal Lack of Lymphangiogenesis Despite Coexpression of VEGF-C and Its Receptor Flt-4 in Uveal Melanoma Invest. Ophthalmol. Vis. Sci., June 1, 2001; 42(7): 1422 - 1428. [Abstract] [Full Text]
|
|
|
|
 |
|
 I. Zachary Signaling mechanisms mediating vascular protective actions of vascular endothelial growth factor Am J Physiol Cell Physiol, June 1, 2001; 280(6): C1375 - C1386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Simons Therapeutic coronary angiogenesis: a fronte praecipitium a tergo lupi? Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H1923 - H1927. [Full Text] [PDF]
|
|
|
|
|
|
S. P. Gunningham, M. J. Currie, C. Han, K. Turner, P. A. E. Scott, B. A. Robinson, A. L. Harris, and S. B. Fox Vascular Endothelial Growth Factor-B and Vascular Endothelial Growth Factor-C Expression in Renal Cell Carcinomas: Regulation by the von Hippel-Lindau Gene and Hypoxia Cancer Res., April 1, 2001; 61(7): 3206 - 3211. [Abstract] [Full Text]
|
|
|
|
|
|
B. Enholm, T. Karpanen, M. Jeltsch, H. Kubo, F. Stenback, R. Prevo, D. G. Jackson, S. Yla-Herttuala, and K. Alitalo Adenoviral Expression of Vascular Endothelial Growth Factor-C Induces Lymphangiogenesis in the Skin Circ. Res., March 30, 2001; 88(6): 623 - 629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Karpanen, M. Egeblad, M. J. Karkkainen, H. Kubo, S. Yla-Herttuala, M. Jaattela, and K. Alitalo Vascular Endothelial Growth Factor C Promotes Tumor Lymphangiogenesis and Intralymphatic Tumor Growth Cancer Res., March 1, 2001; 61(5): 1786 - 1790. [Abstract] [Full Text]
|
|
|
|
|
|
A. Kadambi, C. Mouta Carreira, C.-o. Yun, T. P. Padera, D. E. J. G. J. Dolmans, P. Carmeliet, D. Fukumura, and R. K. Jain Vascular Endothelial Growth Factor (VEGF)-C Differentially Affects Tumor Vascular Function and Leukocyte Recruitment: Role of VEGF-Receptor 2 and Host VEGF-A Cancer Res., March 1, 2001; 61(6): 2404 - 2408. [Abstract] [Full Text]
|
|
|
|
|
|
M. S. Pepper Lymphangiogenesis and Tumor Metastasis: Myth or Reality? Clin. Cancer Res., March 1, 2001; 7(3): 462 - 468. [Abstract] [Full Text]
|
|
|
|
|
|
S. E. Epstein, S. Fuchs, Y. F. Zhou, R. Baffour, and R. Kornowski Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards Cardiovasc Res, February 16, 2001; 49(3): 532 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. E. Hoefer, N. van Royen, I. R. Buschmann, J. J. Piek, and W. Schaper Time course of arteriogenesis following femoral artery occlusion in the rabbit Cardiovasc Res, February 16, 2001; 49(3): 609 - 617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Robinson and S. Stringer The splice variants of vascular endothelial growth factor (VEGF) and their receptors J. Cell Sci., January 3, 2001; 114(5): 853 - 865. [Abstract] [PDF]
|
|
|
|
|
|
R. S. Kiesz, P. Buszman, J. L. Martin, E. Deutsch, M. M. Rozek, E. Gaszewska, M. Rewicki, P. Seweryniak, M. Kosmider, and M. Tendera Local Delivery of Enoxaparin to Decrease Restenosis After Stenting: Results of Initial Multicenter Trial : Polish-American Local Lovenox NIR Assessment Study (The POLONIA Study) Circulation, January 2, 2001; 103(1): 26 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yano, H. Shinohara, R. S. Herbst, H. Kuniyasu, C. D. Bucana, L. M. Ellis, and I. J. Fidler Production of Experimental Malignant Pleural Effusions Is Dependent on Invasion of the Pleura and Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor by Human Lung Cancer Cells Am. J. Pathol., December 1, 2000; 157(6): 1893 - 1903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Gunningham, M. J. Currie, C. Han, B. A. Robinson, P. A. E. Scott, A. L. Harris, and S. B. Fox The Short Form of the Alternatively Spliced flt-4 but not Its Ligand Vascular Endothelial Growth Factor C Is Related to Lymph Node Metastasis in Human Breast Cancers Clin. Cancer Res., November 1, 2000; 6(11): 4278 - 4286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Abraham, R. Hofbauer, R. Schafer, R. Blumer, P. Paulus, A. Miksovsky, H. Traxler, A. Kocher, and S. Aharinejad Selective Downregulation of VEGF-A165, VEGF-R1, and Decreased Capillary Density in Patients With Dilative but Not Ischemic Cardiomyopathy Circ. Res., October 13, 2000; 87(8): 644 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. PARTANEN, J. AROLA, A. SAARISTO, L. JUSSILA, A. ORA, M. MIETTINEN, S. A. STACKER, M. G. ACHEN, and K. ALITALO VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues FASEB J, October 1, 2000; 14(13): 2087 - 2096. [Abstract] [Full Text]
|
|
|
|
|
|
A. Saaristo, T. A. Partanen, J. Arola, L. Jussila, M. Hytonen, A. Makitie, S. Vento, A. Kaipainen, H. Malmberg, and K. Alitalo Vascular Endothelial Growth Factor-C and Its Receptor VEGFR-3 in the Nasal Mucosa and in Nasopharyngeal Tumors Am. J. Pathol., July 1, 2000; 157(1): 7 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Zachary, A. Mathur, S. Yla-Herttuala, and J. Martin Vascular Protection : A Novel Nonangiogenic Cardiovascular Role for Vascular Endothelial Growth Factor Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1512 - 1520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lymboussaki, B. Olofsson, U. Eriksson, and K. Alitalo Vascular Endothelial Growth Factor (VEGF) and VEGF-C Show Overlapping Binding Sites in Embryonic Endothelia and Distinct Sites in Differentiated Adult Endothelia Circ. Res., November 26, 1999; 85(11): 992 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Stacker, K. Stenvers, C. Caesar, A. Vitali, T. Domagala, E. Nice, S. Roufail, R. J. Simpson, R. Moritz, T. Karpanen, et al. Biosynthesis of Vascular Endothelial Growth Factor-D Involves Proteolytic Processing Which Generates Non-covalent Homodimers J. Biol. Chem., November 5, 1999; 274(45): 32127 - 32136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Marchio, L. Primo, M. Pagano, G. Palestro, A. Albini, T. Veikkola, I. Cascone, K. Alitalo, and F. Bussolino Vascular Endothelial Growth Factor-C Stimulates the Migration and Proliferation of Kaposi's Sarcoma Cells J. Biol. Chem., September 24, 1999; 274(39): 27617 - 27622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shizukuda, S. Tang, R. Yokota, and J. A. Ware Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration and Proliferation Depend on a Nitric Oxide–Mediated Decrease in Protein Kinase C{delta} Activity Circ. Res., August 6, 1999; 85(3): 247 - 256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R Kersten, P. S Pagel, W. M Chilian, and D. C Warltier Multifactorial basis for coronary collateralization: a complex adaptive response to ischemia Cardiovasc Res, July 1, 1999; 43(1): 44 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Valtola, P. Salven, P. Heikkila, J. Taipale, H. Joensuu, M. Rehn, T. Pihlajaniemi, H. Weich, R. deWaal, and K. Alitalo VEGFR-3 and Its Ligand VEGF-C Are Associated with Angiogenesis in Breast Cancer Am. J. Pathol., May 1, 1999; 154(5): 1381 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Baldwin, B. Catimel, E. C. Nice, S. Roufail, N. E. Hall, K. L. Stenvers, M. J. Karkkainen, K. Alitalo, S. A. Stacker, and M. G. Achen The Specificity of Receptor Binding by Vascular Endothelial Growth Factor-D Is Different in Mouse and Man J. Biol. Chem., May 25, 2001; 276(22): 19166 - 19171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. FRANCIS, M. K. RAIZADA, A. A. MANGI, L. G. MELO, V. J. DZAU, P. R. VALE, J. M. ISNER, D. W. LOSORDO, J. CHAO, M. J. KATOVICH, et al. Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb? Physiol Genomics, December 21, 2001; 7(2): 79 - 94. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
