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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2573.)
© 2000 American Heart Association, Inc.

Vascular Biology

Coadministration of Angiopoietin-1 and Vascular Endothelial Growth Factor Enhances Collateral Vascularization

Jei Keon Chae; Injune Kim; Seok Tae Lim; Myoung Ja Chung; Won Ho Kim; Hwan Gyu Kim; Jae Ki Ko; Gou Young Koh

From the National Creative Research Initiatives Center for Cardiac Regeneration and Institute of Cardiovascular Research (J.K.C., I.K., H.G.K, J.K.K., G.Y.K.) and the Departments of Nuclear Medicine (S.T.L), Pathology (M.J.C), and Internal Medicine (W.H.K), Chonbuk University Medical School, Chonju, Korea.

Correspondence to Gou Young Koh, MD, PhD, National Creative Research Initiatives Center for Cardiac Regeneration, Chonbuk National University Medical School, San 2-20, Keum-Am-Dong, Chonju, 560-180, Republic of Korea. E-mail gykoh{at}moak.chonbuk.ac.kr

	Abstract

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Abstract—Using growth factors to induce vasculogenesis is a promising approach in the treatment of ischemic legs and myocardium. Because the vasculogenesis requires a cascade of growth factors, their receptors, and intracellular signals, such therapies may require the application of more than a single growth factor. We examined the effect of 2 endothelial cell–specific growth factors, angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), on primary cultured porcine coronary artery endothelial cells. VEGF, but not Ang1, increased DNA synthesis and cell number. Ang1 or VEGF induced migration and sprouting activity, increased plasmin and matrix metalloproteinase-2 secretion, and decreased tissue inhibitors of metalloproteinase type 2 secretion. A combination of the submaximal doses of Ang1 and VEGF enhanced these effects and was more potent than the maximal dose of either alone. In a rabbit ischemic hindlimb model, a combination of Ang1 and VEGF gene delivery produced an enhanced effect on resting and maximal blood flow and capillary formation that was greater than that of either factor alone. Angiographic analyses revealed that larger blood vessels were formed after gene delivery of Ang1 or Ang1 plus VEGF than after VEGF gene delivery. These results suggest that combined treatment of Ang1 and VEGF could be used to produce therapeutic vascularization.

Key Words: angiopoietin-1\b • vascular endothelial growth factor\b • vascularization

	Introduction

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Blood vessels form through 2 distinct processes, vasculogenesis and angiogenesis. In vasculogenesis, endothelial cells differentiate de novo from mesodermal precursors, whereas in angiogenesis, new vessels are generated from preexisting ones.^{1 2 3} In embryos, both processes are essential for normal development. In adults, angiogenesis and neovascularization can be unwanted processes in certain disease states, including tumor growth and diabetic retinopathy. However, formation of new blood vessels can also help alleviate some disease states, as in the formation of collateral circulation in ischemic myocardium.⁴ Indeed, angiogenesis and neovascularization in ischemic myocardium provide an important clinical benefit.⁵

Recently, several studies have examined therapeutic angiogenesis/neovascularization with the use of growth factors as approaches to treat ischemic myocardium.^{5 6 7} However, a single growth factor may be insufficient for therapeutic purposes, because the development of a functional vascular system requires a cascade of growth factors, their receptors, and intracellular signals.^{1 2 3} Of these, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are of particular interest because their receptors are specifically located in endothelial cells.^{8 9} In fact, in a mouse corneal micropocket assay, Ang1 failed to stimulate an angiogenic response when administered alone.¹⁰ However, when coadministered with VEGF, Ang1 augmented postnatal neovascularization.¹⁰ Thus, Ang1, in combination with VEGF, is a candidate for therapeutic vascularization in the treatment of ischemic myocardium.

In the present study, we examined the effect of coadministration of Ang1 and VEGF on cultured coronary artery endothelial cells and the ischemic hindlimb of rabbits. Our results indicate that a combination of Ang1 and VEGF holds promise as a means to produce therapeutic vascularization.

	Methods

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Materials and Cell Culture
We obtained recombinant Ang1 protein, Ang1*, from Regeneron Pharmaceuticals, Inc. Recombinant human VEGF₁₆₅ was purchased from R&D Systems. Primary cultured porcine coronary artery endothelial cells (PCAECs) were prepared and maintained as previously described.¹¹ The primary cultured cells used in the present study were between passages 2 and 4.

Measurements of DNA Synthesis, Cell Number, Migration, and Sprouting Activity
The amount of DNA was measured with PicoGreen fluorescent reagent (Molecular Probes) as described by Singer et al.¹² Cells were counted with a Coulter Counter System after trypsinization. Migration and sprouting assays were performed with the use of microcarrier beads as previously described.¹³

Measurements of Plasmin, MMP-2, and TIMP2
Measurements of plasmin, matrix metalloproteinase (MMP)-2, and tissue inhibitors of metalloproteinase (TIMP)2 in culture medium were performed as previously described.¹³

Plasmids
Human Ang1 or VEGF₁₆₅ cDNA was inserted into the cytomegalovirus promoter–driven mammalian cell expression vector, pcDNA3.1/myc-His (Invitrogen). The viral inverted terminal repeat sequences from adeno-associated virus can increase transgene expression,¹⁴ so the inserts were flanked by 2 copies of the right inverted terminal repeat sequence of the adeno-associated virus on each side (please see Figure I, published online at http://atvb.ahajournals.org).

Animal Model
New Zealand White rabbits (male, mean age 12 to 14 months, 3.8 to 4.2 kg) were used for the present study. The hindlimb ischemic model was produced according to Takeshita et al.¹⁵ The schematic procedures are shown (please see Figure I, published online at http://atvb.ahajournals.org).

Intramuscular Gene Transfer
At day 10, plasmid DNAs were injected directly with a 25-gauge needle into the 3 major thigh muscles of the ischemic hindlimb. For each rabbit, 125 µg of plasmid control (pControl, n=7), plasmid VEGF (pVEGF, n=8), or plasmid Ang1 (pAng1, n=8) per 0.5 mL of normal saline was injected at each of 4 sites (total, 500 µg/2.0 mL). For combined gene transfer (n=8), 0.5 mL of normal saline containing 125 µg of pVEGF and 125 µg of pAng1 was injected.

Selective Angiography
Selective internal iliac angiography was performed with a hand injection of 5 mL of nonionic contrast medium at a flow rate of 1 mL/s. Vascularization of the left thigh was quantified by the method of Pu et al.¹⁶

In Vivo Resting and Maximum Blood Flow Measurements
Blood flow was measured by using a 0.014-in Doppler guidewire (Cardiometrics) according to the methods of Bauters et al¹⁷ at rest and after bolus injection of 5 mg papaverine.

Capillary Density
Tissue sections were prepared from adductor muscles of the ischemic hindlimbs at day 40. Capillary density was calculated by counting numbers of capillaries per 1000 myocytes after staining the capillary endothelial alkaline phosphatase with an indoxyl-tetrazolium.

Human VEGF and Ang1 Gene Expression in Ischemic Skeletal Muscle
Gene expression was evaluated by reverse transcription–polymerase chain reaction in the tissues of injected areas and other tissues from 9 additional rabbits with hindlimb ischemia killed at 3, 7, and 30 days after gene transfer (n=3 at each time point).

Statistical Analysis
Data are expressed as mean±SD. Statistical significance was tested by t test or 1-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05.

An expanded Methods section can be found in an online data supplement available at http://atvb.ahajournals.org.

	Results

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VEGF, but Not Ang1, Increases DNA Synthesis and Cell Number in PCAECs
Consistent with previous reports with other types of endothelial cells,⁸ VEGF increased DNA synthesis and cell number in a dose-dependent manner in PCAECs (please see Figure II, published online at http://atvb.ahajournals.org). However, Ang1 (50 to 400 ng/mL) did not produce any changes in DNA synthesis or cell number compared with control (please see Figure II, published online at http://atvb.ahajournals.org). The dose of VEGF (10 ng/mL) showing submaximal effect was chosen for use in combination with Ang1 (200 ng/mL). The combined treatment of Ang1 and VEGF produced an enhanced effect on DNA synthesis and cell number (please see Figure II, published online at http://atvb.ahajournals.org).

Combined Treatment With Ang1 and VEGF Enhances Migration and Sprouting in PCAECs
Placing microcarrier beads onto a confluent monolayer of PCAECs for 2 to 3 days produces beads covered by a confluent monolayer of cells with 25 to 30 cells per bead. When PCAEC-bearing microcarrier beads were placed onto gelatinized plastic dishes with control buffer for 20 hours, they yielded a basal level of nondirectional migration (55 to 60 cells per 10 beads, Figure 1; please see Figure III, published online at http://atvb.ahajournals.org). The number of migrating cells increased with Ang1 or VEGF stimulation in a dose-dependent manner. The migration potency of Ang1 was less than that of VEGF. The PCAEC-bearing microcarrier beads were embedded in 3D fibrin gels and cultured. Daily addition of growth factors increased sprout formation in a dose-dependent manner (Figure 2; please see Figure IV, published online at http://atvb.ahajournals.org). The sprouting potency of Ang1 was also less than that of VEGF. The doses of Ang1 (200 ng/mL) and VEGF (10 ng/mL) showing a submaximal effect on sprouting activity were used in combination. The combination produced an enhanced effect on migration and sprout formation (Figures 1 and 2; please see Figures III and IV, published online at http://atvb.ahajournals.org).

View larger version (28K): [in this window] [in a new window]   Figure 1. . Effect of single and combined treatment with Ang1 and VEGF on migratory in cultured PCAECs. To measure migratory activity, the cells were grown to confluence on microcarrier beads, and the beads were placed in gelatinized 24-well plates in medium containing 2% serum and various amounts of recombinant proteins and incubated for 20 hours. The number of endothelial cells that migrated from the beads and attached to the well per 10 beads was counted. Bars represent mean±SD from 4 experiments. A+V indicates combination of Ang1 (200 ng/mL) and VEGF (10 ng/mL). *P<0.05 vs control buffer; #P<0.05 vs Ang1 (400 ng/mL); and P<0.05 vs VEGF (30 ng/mL).

View larger version (29K): [in this window] [in a new window]   Figure 2. . Effect of single and combined treatment with Ang1 and VEGF on sprouting activity in cultured PCAECs. To measure sprouting activity, cells grown on microcarrier beads were placed in fibrin gels containing growth factors and were incubated with daily supplementation with the same amount of growth factors. After 3 days, the extent of sprouting was examined with an inverted phase-contrast microscope. The number of endothelial sprouts with length exceeding the bead diameter (175 µm) per 40 microcarrier beads was counted. Bars represent mean±SD from 4 experiments. *P<0.05 vs control buffer; #P<0.05 vs Ang1 (300 ng/mL); and P<0.05 vs VEGF (30 ng/mL).

Combined Treatment With Ang1 and VEGF Enhances Plasmin and MMP-2 Secretion but Suppresses TIMP2 Secretion From PCAECs
Using the submaximal doses defined above, we assessed the effect of the growth factors on the secretory activity of cells. Compared with the addition of control buffer, Ang1 or VEGF produced 2.5-fold or 3.5-fold increases, respectively, in plasmin secretion for 3 hours (please see Figure V, published online at http://atvb.ahajournals.org). Plasmin secretion was confirmed by fibrin zymography (Figure 3). Compared with cells treated with buffer alone, culture medium from Ang1 or VEGF clearly increased the 85-kDa fibrinolytic bands (Figure 3; please see Figure VI, published online at http://atvb.ahajournals.org). Compared with the addition of control buffer, addition of Ang1 or VEGF produced 2.0-fold or 2.8-fold increases, respectively, in MMP-2 secretion for 3 hours (please see Figure V, published online at http://atvb.ahajournals.org). The MMP-2 secretion was confirmed by gelatin zymography (Figure 3). Compared with the cells treated with buffer alone, culture medium from Ang1 or VEGF clearly increased the 68-kDa gelatinolytic bands (pro form of MMP-2, Figure 3; please see Figure VI, published online at http://atvb.ahajournals.org). However, no marked 62-kDa gelatinolytic bands (active form of MMP-2) were observed from any treatment. Addition of Ang1 or VEGF suppressed the basal secretion of TIMP2 by 45% or 49%, respectively (please see Figure V, published online at http://atvb.ahajournals.org). Combination treatment of Ang1 and VEGF produced an enhanced effect on the induction of plasmin and MMP-2 secretion and on the suppression of TIMP2 secretion that was greater than the effect of either agent alone (Figure 3; please see Figures V and VI, published online at http://atvb.ahajournals.org).

View larger version (61K): [in this window] [in a new window]   Figure 3. . Effect of single and combined treatment of Ang1 and VEGF on secretion of plasmin and MMP-2 in cultured PCAECs. Zymographic analysis of the secretion of plasmin and MMP-2 in culture medium of PCAECs is shown. Cells were incubated in serum-free and phenol red–free DMEM for 24 hours. Control buffer (CO), Ang1 (AP, 200 ng/mL), VEGF (VE, 10 ng/mL), or a combination of the 2 growth factors (AV) was added to 0.5 mL of culture medium and incubated for the times indicated. Five microliters of 10-fold concentrated sample were loaded on gels for fibrin zymography (top) and gelatin zymography (bottom). Lane S contains standards of plasmin, the active form of MMP-9 (aMMP9), the pro form of MMP-2 (pMMP2), and the active form of MMP-2 (aMMP2). Molecular weight markers (open arrowheads at right) were used to estimate molecular masses.

Combined Gene Transfer of Ang1 and VEGF Enhances Angiographic Collateral Vessels in the Ischemic Hindlimb of Rabbits
Representative angiograms were recorded from each group at day 40. The pAng1, pVEGF, and pAng1+pVEGF limbs contained more prominent blood vessels, particularly corkscrew-shaped collateral vessels in the midzone region, than did the control limbs (Figure 4). In all cases, more and larger vessels were noted in the pAng1 limbs compared with the pVEGF limbs. In the limbs with combined gene transfer, more prominent collateral vessels were formed (Figure 4). Quantitative assay revealed no significant difference in angiographic vessel count at the midthigh of the ischemic hindlimb among the groups on day 10 (Figure 5). At day 40, angiographic vessel count in pControl limbs had not markedly changed, whereas in pAng1, pVEGF, and pAng1+pVEGF limbs, the angiographic vessel count increased 2.0-fold (Figure 5). Although there were no statistically significant differences among the 3 groups, more vessel numbers were formed in the order pAng1+pVEGF>pVEGF>pAng1.

View larger version (161K): [in this window] [in a new window]   Figure 4. . Effect of single and combined gene transfer of Ang1 and VEGF on internal iliac artery angiographic findings in ischemic hindlimbs of rabbits. Representative internal iliac artery angiograms in rabbits at day 40 after gene transfer are shown. Direct intramuscular injection of 500 µg of pControl (A), pAng1 (B), pVEGF (C), or pAng1+pVEGF (D) was performed.

View larger version (19K): [in this window] [in a new window]   Figure 5. . Effect of single and combined gene transfer of Ang1 and VEGF on angiographic vessel counts in ischemic hindlimbs of rabbits. Angiographic vessels were counted before (day 10) and 30 days after (day 40) direct intramuscular gene transfer of 500 µg of pControl (A), pAng1 (B), pVEGF (C), or pAng1+pVEGF (D). Vascularization of the left thigh was quantified by directly counting the number of vessels crossing a line drawn vertically across the midthigh portion (passing through the center of the femur) on the film taken 4 seconds after injection of contrast in each series of angiograms. Data represent mean±SD. *P<0.01 vs day 10; #P<0.05 vs pControl.

Combined Gene Transfer of Ang1 and VEGF Enhances Blood Flow in the Ischemic Hindlimb of Rabbits
At day 10, the resting and maximal blood flows were similar for all groups (please see Figure VII, published online at http://atvb.ahajournals.org). The resting blood flow was higher in pAng1 and pVEGF compared with pControl at days 40 and 70, although there were no statistically significant differences (please see Figure VII, published online at http://atvb.ahajournals.org). However, the resting blood flow in the pAng1+pVEGF group was significantly higher than in the pControl group (please see Figure VII, published online at http://atvb.ahajournals.org). The maximal blood flows in pAng1, pVEGF, and pAng1+pVEGF groups were higher than in the pControl group at days 40 and 70 (please see Figure VII, published online at http://atvb.ahajournals.org). Notably, at day 70, the combined group had almost same maximal blood flow and had higher maximal blood flow than did the pAng1 group or pVEGF group alone (please see Figure VII, published online at http://atvb.ahajournals.org).

Single or Combined Gene Transfer of Ang1 and VEGF Increases Capillary Density in the Ischemic Hindlimb of Rabbits
At day 40, the number of capillaries per 1000 myocytes was greater in pAng1, pVEGF, and pAng1+pVEGF groups than in the pControl group (Figure 6; please see Figure VIII, published online at http://atvb.ahajournals.org). Notably, the combined group had a higher capillary density than was found for the pAng1 group or pVEGF group alone (Figure 6; please see Figure VIII, published online at http://atvb.ahajournals.org).

View larger version (36K): [in this window] [in a new window]   Figure 6. . Effect of gene transfer on capillary density in ischemic hindlimbs of rabbits. Tissue sections were obtained from ischemic hindlimbs at day 40 and were stained for alkaline phosphatase, postfixed, and counterstained with eosin. Capillary densities were measured as described in Methods. Data represent mean±SD. pA+V indicates pAng1+pVEGF. *P<0.01 vs pControl; #P<0.01 vs pVEGF.

Human Ang1 and Ang2 Gene Expression in Ischemic Skeletal Muscle
The expression of exogenous human VEGF and Ang1 mRNAs in sections of semimembranous muscles was examined at days 0, 3, 7, and 30 after gene transfer into ischemic rabbit hindlimbs. The expression of both genes was maximal at day 3, dramatically decreased at day 7, and not detectable at day 30 (please see Figure IX, published online at http://atvb. ahajournals.org). Thus, the expression of both genes was transient. In the combined gene transfer, the 2 human mRNAs were expressed at almost equal levels at each time point (please see Figure IX, published online at http://atvb.ahajournals.org). No human Ang1 or VEGF mRNA was detected in tissue samples from the distal ischemic hindlimb or sites remote from the ischemic hindlimb, including the heart, lung, liver, and semimembranous muscle of the contralateral limb (data not shown).

	Discussion

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Therapeutic vascularization using either gene or peptide delivery of growth factors holds great promise for treating ischemic disease.^{5 6 7} To enhance vascularization, endothelial cells need to increase their proliferation, migration, and sprouting. Our results indicate that VEGF, but not Ang1, is a strong mitogen for coronary artery endothelial cells. In addition, both Ang1 and VEGF have the ability to induce migration, sprouting activity, and secretion of plasmin and MMP-2 and to reduce TIMP2 secretion in coronary artery endothelial cells. VEGF is more potent than Ang1. However, our notable finding is that a combination of submaximal doses of Ang1 and VEGF produces an enhanced effect on these cellular mechanisms that is more potent than the maximal dose of VEGF alone. Indeed, in the ischemic hindlimb of rabbits, our combination of Ang1 and VEGF gene delivery produced an enhanced effect on blood flow and capillary formation that was more than the effect of Ang1 or VEGF alone. Although the gene expression in the ischemic muscles after single and direct gene delivery was transient (up to 7 days), it should be noted that the limbs showed functional and histological improvements. And, in a clinical setting, transient expression may actually be desirable because long-term expression may produce unwanted side effects. Therefore, we conclude that a combined gene delivery of Ang1 and VEGF can be useful for enhancing therapeutic vascularization in vivo.

The molecular and cellular effects of VEGF on endothelial cells are well known,^{5 18} whereas those of Ang1 are less well known. Ang1 has been identified as a ligand of the endothelial cell–specific Tie2 receptor.⁸ In vivo analyses by targeted gene inactivation have suggested that Ang1 recruits and sustains periendothelial support cells for vessel formation. In vitro experiments have shown that Ang1 has specific effects on endothelial cells: it potently induces chemotactic response,¹⁹ network formation,²⁰ sprouting,^{13 21} and survival in apoptosis,^{20 22} but it does not have mitotic effects.^{8 23} The present data indicate that Ang1 is a potent factor for migration and sprouting in coronary endothelial cells and have confirmed its lack of mitotic effect. These observations are consistent with studies of Ang1 in other types of endothelial cells.^{13 19 21 23}

To migrate and sprout in vivo, endothelial cells secrete proteinases to dissolve the adjacent extracellular matrix.²⁴ One family of such proteinases is the MMPs. There are currently 20 known members of the MMPs. In addition, 4 members of the TIMPs have been identified to date.²⁵ Vascular endothelial cells secrete mainly pro-MMP-2 and TIMP2.^{13 24} TIMP2 has a dual role in its interaction with pro-MMP2. Interaction of TIMP2 with pro-MMP-2 and membrane type MMP-1 facilitates cell surface–mediated activation, whereas interaction with active MMP-2 results in inhibition.²⁵ Thus, an increase in the ratio of active MMP-2 to TIMP2 favors the degradation of matrix proteins to enhance vascularization. Our in vitro results indicate that Ang1 and VEGF increase pro-MMP-2 (but not active MMP-2) secretion and decrease TIMP2 secretion from coronary endothelial cells. However, our in vivo results indicate that Ang1 and VEGF produce increased vascularization through degradation of adjacent matrix proteins. Therefore, how Ang1 and VEGF are involved in increasing the ratio of active MMP-2 to TIMP2 in vivo will be examined in future studies.

The VEGF gene is already in the clinical trials for therapeutic vascularization,²⁶ whereas the Ang1 gene is still being tested in animals.^{27 28} Consistent with previous reports,^{28 29} our results indicate that gene transfer of VEGF or Ang1 increased vascularization in the ischemic hindlimb of rabbits. Although the angiographic vessel count and capillary density after Ang1 gene transfer were less than that from VEGF gene transfer, the resting and maximal blood flows were similar between the 2 groups, possibly because thicker vessels formed in response to Ang1. Therefore, in future studies, we will examine the mechanism of how Ang1 produces new vessels in vivo. As we predicted, on the basis of the in vitro effects, our in vivo results indicated that combined gene transfer of Ang1 and VEGF produced more increased collateral circulation, resting and maximal blood flow, and capillary density. Therefore, for efficient and potent vascularization, the combined gene transfer of Ang1 and VEGF could be a valuable tool.

Although VEGF is known to be a powerful growth factor for therapeutic angiogenesis/vascularization in the ischemic hindlimb and myocardium, it has other activities that can increase the proliferation and permeability of capillary endothelial cells. These activities may produce unwanted side effects, such as tumor angiogenesis, vascular leakage, edema, and inflammation.^{7 30} Although Ang1 has no proliferative effect on endothelial cells, interestingly, transgenic overexpression or gene transfer of Ang1 increases vascularization.^{27 28} Furthermore, a recent report indicates that overexpression of VEGF in the skin produced leaky blood vessels, whereas overexpression of Ang1 produced nonleaky blood vessels.³¹ Importantly, the combined overexpression of Ang1 and VEGF had an additive effect on neovascularization but produced the leakage-resistant vessels typical of Ang1 overexpression.³¹ Moreover, systemic delivery of Ang1 by adenoviral gene delivery causes resistance to vascular leakage induced by mustard oil and VEGF in adult vessels.³² Thus, Ang1, in combination with VEGF, is a promising and safe candidate for therapeutic vascularization in the treatment of ischemic myocardium.

In summary, the present study provides further insight in the cellular mechanism of the 2 endothelial cell–specific growth factors, Ang1 and VEGF. In addition, our results indicate that a combination of Ang1 and VEGF could be better than either single growth factor for enhancing therapeutic vascularization in vivo.

	Acknowledgments

 
This work was supported by the Creative Research Initiatives of the Korean Ministry of Science and Technology and by the Research Foundation and Post-Doc Program (1999) from Chonbuk National University.

Received March 6, 2000; accepted August 25, 2000.

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