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. 1998 Aug;153(2):381-94.
doi: 10.1016/S0002-9440(10)65582-4.

Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia

B Witzenbichler  1 , T Asahara, T Murohara, M Silver, I Spyridopoulos, M Magner, N Principe, M Kearney, J S Hu, J M Isner
Affiliations

Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia

B Witzenbichler et al. Am J Pathol. 1998 Aug.

Abstract

Recently, vascular endothelial growth factor-C (VEGF-C or VEGF-2) was described as a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3. In vivo data, limited to constitutive overexpression in transgenic mice, have been interpreted as evidence that the growth-promoting effects of VEGF-C are restricted to development of the lymphatic vasculature. The current studies were designed to test the hypothesis that constitutive expression of VEGF-C in adult animals promotes angiogenesis. In vitro, VEGF-C exhibited a dose-dependent mitogenic and chemotactic effect on endothelial cells, particularly for microvascular endothelial cells (72% and 95% potency, respectively, compared with VEGF-A/VEGF-1). VEGF-C stimulated release of nitric oxide from endothelial cells and increased vascular permeability in the Miles assay; the latter effect was attenuated by pretreatment with the nitric oxide synthase inhibitor N(omega)-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 microg) from the polymer coating of an angioplasty balloon (n = 8 each) or as recombinant human protein (rhVEGF-C; 500 microg) 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.

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Figures

Figure 1.
Figure 1.
Proliferative response of HUVECs (A) or HMECs (B) to rhVEGF-A/VEGF-1 or VEGF-C/VEGF-2 protein. Cells (5 × 103) were seeded per well, and increase in cell number was assessed 48 hours after addition of growth factors using the MTS-colorimetric assay. Data are means (bars, SEM) of parallel samples. *P < 0.05 VEGF-A/VEGF-1 versus VEGF-C/VEGF-2 protein.
Figure 2.
Figure 2.
Migratory response of HUVECs (A) or HMECs (B) to rhVEGF-A/VEGF-1 or VEGF-C/VEGF-2 protein. Cells (2.5 × 104) were seeded in the upper wells of a 48-well microchemotaxis Boyden chamber and incubated for 4 hours at 37°C in medium 199 supplemented with 1% FBS. The lower wells contained different concentrations of growth factor. Cells migrating through a polycarbonate membrane with a pore size of 8 μm were quantified by staining the cells at the lower side of the membrane with Giemsa solution and counting three high-power fields (×100). Each condition was done in quadruplicate. Data are means (bars, SEM) of parallel samples. *P < 0.05 VEGF-A/VEGF-1 versus VEGF-C/VEGF-2 protein.
Figure 3.
Figure 3.
Induction of NO release from HUVECs by VEGF-C/VEGF-2, measured with an NO-specific polarographic electrode connected to an NO meter. HUVECs in six-well plates were bathed with Krebs-Henseleit solution before addition of reagents. Baseline is defined as baseline fluctuation over a period of 5 minutes of NO production by cells not exposed to agonist. The NO donor S-nitroso-N-acetyl-penicillamine (0.1 mmol/L) and VEGF-A/VEGF-1 (100 ng/ml) were used as positive controls. Administration of the NO inhibitor l-NAME abrogated the effect of VEGF-C and served as a negative control. Data shown are means (bars, SEM) of four to six measurements in each group. *P < 0.05 versus 100 ng/ml VEGF-C.
Figure 4.
Figure 4.
Effect of VEGF-A and VEGF-C on vascular permeability (Miles assay). A: VEGF-A as well as VEGF-C increased vascular permeability in a dose-dependent manner. Doses are indicated by the arrows. A small area of traumatic bluing was observed after injection of saline control. B: Effect of the NO synthase inhibitor l-NAME (20 mg/kg) on VEGF-A- and VEGF-C-induced vascular permeability. Systemic injection of l-NAME 20 minutes before administration of Evans blue dye attenuated VEGF-A- and VEGF-C-mediated vascular permeability. Photographs are representative of three experiments each.
Figure 5.
Figure 5.
Reverse transcription-PCR demonstrates VEGFR-1, VEGFR-2, and VEGFR-3 mRNA expression in explanted segments of normal human saphenous vein (lane 1) and internal mammary artery (lane 2). HUVECs (lane 3), known to coexpress all three receptors, served as a positive control, whereas cultured human fibroblasts (lane 4) served as a negative control. M: DNA size marker. Sizes of the PCR products are indicated.
Figure 6.
Figure 6.
A: Ratio of hindlimb perfusion pressures at day 0 (immediately before treatment) and day 30. The blood pressure ratio was defined for each rabbit as the ratio of systolic pressure of the ischemic limb to systolic pressure of the normal limb (n = 8 each). At day 0, no differences were observed among the groups. At day 30, the blood pressure ratio is significantly greater in rabbits receiving rhVEGF-C/VEGF-2 protein versus RSA controls and in rabbits receiving pcVEGF-C/VEGF-2 plasmid versus pGSVLacZ controls. B: Angiographic score, derived by quantitative analysis of angiographically demonstrable vessels in the medial thigh of the ischemic hindlimb, at days 0 and 30. At day 0, there are no differences among the groups. At day 30, the number of vessels is significantly greater in the VEGF-C/VEGF-2 protein- and plasmid-treated groups compared with controls. Administration of plasmid DNA appeared to yield a more pronounced effect than use of recombinant protein (n = 8 each). C: Iliac blood flow reserve (ratio between blood flow at rest and maximal flow induced by nitroprusside) measured from the internal iliac artery of the ischemic limb at day 30, using intra-arterial Doppler wire. Both VEGF-C/VEGF-2 protein and plasmid significantly increased iliac flow reserve (n = 8 each). D: Capillary density evaluated at day 30 in histological sections harvested from the medial thigh muscles of the nonischemic and ischemic limbs. In the nonischemic limb, capillary density was not different among groups (not shown). In the ischemic limb, capillary density is increased significantly by VEGF-C/VEGF-2 protein and plasmid (n = 8 each). NS, not significant.
Figure 7.
Figure 7.
Selective internal iliac angiography at day 30 of animals receiving RSA control (A), rhVEGF-C/VEGF-2 protein (B), pGSVLacZ control plasmid (B), and pcVEGF-C plasmid DNA (D). In contrast to controls, treatment groups exhibited an increase in angiographically visible collateral blood vessels. (Ruler cm marking.)
Figure 8.
Figure 8.
Histological sections retrieved at day 30 from ischemic hindlimb muscle (musculus adductor) and stained with alkaline phosphatase (counterstained with eosin). A: RSA control; B: rhVEGF-C/VEGF-2 protein; C: pGSVLacZ control plasmid; D: pcVEGF-C/VEGF-2 plasmid-treated rabbit. Administration of VEGF-C as recombinant protein or plasmid resulted in increased capillary density. Dots, which appear dark blue, indicate capillaries.
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