Vascular Endothelial Growth Factor165 Gene Transfer Augments Circulating Endothelial Progenitor Cells in Human Subjects
Abstract—Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%, P<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector, P<0.001; VEGF versus saline, P<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, αvβ3, and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.
Recent investigations have established the feasibility of using recombinant formulations or gene transfer of angiogenic growth factors to expedite and augment collateral artery development in patients with tissue ischemia.1 2 3 4 Such postnatal neovascularization was initially considered synonymous with proliferation and migration of preexisting, fully differentiated endothelial cells (ECs) resident within parent vessels, ie, angiogenesis.5 6 The demonstration, however, of postnatal circulating bone marrow–derived endothelial progenitor cells (EPCs) that may home to sites of neovascularization and differentiate into ECs in situ7 is consistent with “vasculogenesis,”6 a critical paradigm for establishment of the primordial vascular network in the embryo. Although the proportional contributions of angiogenesis and vasculogenesis to neovascularization of adult organisms remain to be clarified, the notion that growth and development of new blood vessels in the adult is not restricted to angiogenesis but encompasses both embryonic mechanisms has now been verified by several laboratories.8 9 10
Among the mechanisms that may modulate the contribution of vasculogenesis to postnatal neovascularization, we considered that certain angiogenic growth factors, which are acknowledged to promote both angiogenesis and vasculogenesis in the embryo11 but have been assumed to promote neovascularization exclusively by angiogenesis in the adult,12 13 14 may in fact promote migration, proliferation, and mobilization of EPCs from adult bone marrow. Indeed, investigations performed in our laboratory using a bone marrow transplant model established that vascular endothelial growth factor (VEGF) may mobilize EPCs from murine bone marrow, resulting in augmented neovascularization.15
Accordingly, the current study was designed to test the hypothesis that intramuscular gene transfer of naked plasmid DNA encoding human VEGF (phVEGF165) may increase the number of circulating human EPCs in patients with critical limb ischemia.
Materials and Methods
The potential for VEGF to enhance the population of circulating EPCs was serially monitored in 20 patients (11 women and 9 men, age 59±16 years) undergoing intramuscular phVEGF165 gene transfer for critical limb ischemia. The same studies were performed on 9 patients who did not receive phVEGF165 gene therapy. Four of these were healthy volunteers (4 men) ranging in age from 30 to 43 years (36±3), who were injected with normal saline. The remaining 5 patients included 2 women and 3 men with critical limb ischemia ages 48 to 78 years (67±12), who had been randomly assigned to receive a control (empty) vector.
Intramuscular phVEGF165 Transfer
The patients undergoing gene therapy received the eukaryotic expression vector pUC118 encoding VEGF165 transcriptionally regulated by the cytomegalovirus promoter/enhancer. A total of 4000 μg of DNA in 8 aliquots of 2.5 mL of sterile saline was administered at different sites into the ischemic limb by direct intramuscular injection.
Plasma VEGF Levels
Plasma levels of VEGF were measured by an ELISA assay in patients before intramuscular injection and weekly up to 4 weeks after the initial set of injections.3
Isolation of Mononuclear Cells (MNCs)
Blood samples were obtained from all individuals before and, weekly, up to 4 weeks after intramuscular injections. Peripheral blood MNCs were isolated in a cell preparation tube by density gradient centrifugation.
EPC Culture Assay
The culture system used in our laboratory to quantify circulating EPCs has been described elsewhere.15 MNCs from 500 μL of peripheral blood were cultured in EC basal medium-2 supplemented with endothelial cell growth medium (EGM-2) microvascular (MV) Single Quots. After 4 days, fluorescence staining of adherent cells was used to detect the binding of Ulex europaeus agglutinin I (UEA-1) and the uptake of acetylated LDL (acLDL). Dual-staining cells positive for both FITC-labeled UEA-1 and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)–labeled acLDL were judged as EPCs and counted per well.
Flow Cytometry Analysis
A total of 2 to 3×105 cells were incubated for 30 minutes at 4°C with monoclonal antibodies prepared against KDR, VE-cadherin, CD62E (E-selectin), CD51/61 (αvβ3), CD31, CD34, and CD14. Quantitative fluorescence-activated cell sorter (FACS) analysis was performed on a FACStar flow cytometer.
All results are expressed as mean±SEM. Statistical significance was evaluated using unpaired Student t test and ANOVA. A value of P<0.05 was interpreted to denote statistical significance. The relationship between variables was determined by linear regression analysis.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
To evaluate the effect of VEGF on EPC kinetics, we obtained 100 samples of peripheral blood from patients with critical limb ischemia undergoing VEGF165 gene transfer. A total of 25 samples were obtained from patients in whom injection of empty DNA vector was performed. An additional 20 samples were obtained from healthy individuals who received saline injections.
Transgene Expression After VEGF Gene Therapy
Plasma levels of VEGF transiently increased in all patients after gene transfer. A mean 2.1±0.3-fold increase over baseline at day 7 (P<0.001) was followed by persistent elevation at day 14 (1.9±0.3-fold, P<0.001), day 21 (1.8±0.2-fold, P<0.01), and day 28 (1.5±0.2-fold, P<0.05, all versus baseline). The mean plasma VEGF concentrations increased from 33.1±5 pg/mL at baseline to a maximum value of 98.8±17 pg/mL after gene transfer (P<0.01). In contrast, no significant changes were documented in the control patients (Figure 1⇓).
EPC Culture Assay After phVEGF165 Gene Therapy
Two independent criteria were used to assess the effect of phVEGF165 gene transfer on EPC kinetics. First, we applied a previously described15 culture assay in which EPCs were quantified by identification of cultured cells demonstrating both UEA-1 reactivity and uptake of acLDL (Figure 2⇓). The impact of VEGF on circulating EPCs could be detected by culture assay from day 7 (80% increase versus baseline, P<0.0005) through day 14 (154% increase versus baseline, P<0.0005), day 21 (82% increase versus baseline, P<0.05), and day 28 after treatment (153% increase versus baseline, P<0.005). The increase in EPCs correlated with the rise in VEGF plasma levels (R2=0.83; P<0.0001) (Figure 1B⇑ online; available at http://www.circresaha.org). Patients injected with empty vector had comparable numbers of EPCs at baseline but failed to exhibit a significant change in cultured EPCs. The number of cultured EPCs was significantly different between the VEGF-treated patients and the control vector–injected group as early as day 7 (P<0.001) and over the following 3 weeks (day 14, P<0.005; day 21, P<0.05; day 28, P<0.005). Likewise, the saline-injected group showed no significant change in EPC counts over 4 weeks after gene transfer. There was no significant difference in EPC count between the saline- and empty vector–treated groups (Figure 1⇑).
FACS analysis was used as a second independent measure to quantify the population of EPCs mobilized in response to VEGF gene transfer. Overall, we observed an average increase in the expression levels of the EC-specific antigens KDR (22.1±1.5-fold versus baseline, P<0.005), VE-cadherin (26±2-fold versus baseline, P<0.001), and CD34 (8±1.5-fold versus baseline, P<0.01). The number of KDR- and VE-cadherin–positive cells increased significantly over baseline values (KDR, 5.6±0.8×103/mL; VE-cadherin, 4.5±1.2×103/mL) as early as day 7 (KDR, 131.2±36 103/mL [P<0.005]; VE-cadherin, 134±45×103/mL [P<0.01]) and continued to be elevated over the entire observation period (day 14, KDR, 112±35 103/mL [P<0.01], and VE-cadherin, 103±19×103/mL [P<0.0005]; day 21, KDR, 106.4±32 103/mL [P<0.01], and VE-cadherin, 105±26×103/mL [P<0.005]; day 28, KDR, 148.9±48 103/mL [P<0.05], and VE-cadherin, 132.6±38×103/mL, [P<0.02]) (Figure 3⇓). In contrast, no significant changes were observed in the group injected with empty plasmid or in the group of healthy volunteers injected with saline. Likewise, the number of CD14-positive cells remained unchanged in all 3 study groups (data not shown). Representative of the measurements taken at the aforementioned time points, Figure 4⇓ shows the expression values at day 14. The increase over baseline in VEGF plasma levels and in VE-cadherin–positive cells showed a high correlation at this time point (R2=0.82; P<0.0001) (Figure 1C⇑ online; available at http://www.circresaha.org). Findings in the VEGF-treated group differed significantly from both control groups.
To determine the relationship between the culture assay and the FACS analysis, we compared the individual increases in both assays at a representative time point (day 14), using the rise in the EPC count and in the number of VE-cadherin positive cells. The results revealed a positive correlation (R2=0.77; P<0.0005) (Figure 1A⇑ online; available at http://www.circresaha.org).
Expression of EC Adhesion Molecules
We also examined the effect of VEGF gene transfer on the expression of EC adhesion molecules. In patients subjected to VEGF gene transfer, the number of circulating MNCs with the surface expression of αvβ3 (CD51/61) and E-selectin (CD62E) increased during the observation period on average by 5±1-fold and 25±1-fold, respectively. In contrast, expression values in individuals receiving empty vector and saline treatment remained unchanged. The expression pattern and levels of E-selectin were very similar to those of VE-cadherin and KDR, with maximal values at day 7 and day 28 after the treatment, whereas the levels for αvβ3 were slightly different with a maximal expression at day 14. At the representative time point, day 14, levels of αvβ3 and E-selectin expression in the VEGF-treated group increased significantly compared with baseline (for αvβ3 [CD51/61], 16.7±6 versus 1.9±0.7×103/mL [P<0.05], and for E-selectin [CD62E], 101±37 versus 3.6±1×103/mL [P<0.02]) and compared with both control groups (for αvβ3 [CD51/61], VEGF versus empty plasmid, P<0.05, and VEGF versus saline, P<0.05; for E-selectin [CD62E], VEGF versus empty plasmid, P<0.02, and VEGF versus saline, P<0.02) (Figure 4⇑).
Preclinical studies in animal models16 and early studies performed in small numbers of patients with lower limb17 and myocardial2 4 ischemia support the notion that gene transfer of VEGF DNA may promote neovascularization of ischemic tissues. These previous reports established that direct injection of phVEGF165 into muscle of the ischemic limb,3 17 as well as into ischemic myocardium,2 transiently elevates plasma VEGF levels in the systemic circulation, a finding that is confirmed in the patients described above.
The current series of patients further establishes that the rise in plasma levels of VEGF is associated with modulation of EPC kinetics after VEGF gene transfer. The increase in EPCs was statistically significant as early as 1 week after gene transfer and remained statistically significant at 2, 3, and 4 weeks follow-up. By comparison, EPC kinetics in the control subjects—including patients with or without critical limb ischemia, injected with empty vector or saline—were unchanged.
Because of limitations in the types of analyses that may be performed in human subjects, the origin and fate of the augmented population of circulating EPCs in these patients must be inferred from experiments performed previously in live animal models. Daily intraperitoneal injection of recombinant human VEGF165 (rhVEGF) to C57BL/6J mice for 1 week increased the total number of circulating EPCs.15 These effects were abrogated by coincidental application of a neutralizing antibody prepared against rhVEGF.
When mice were pretreated with rhVEGF or control buffer for 7 days before cornea micropocket injury and then examined on day 7 after injury (ie, 7 days after the last dose of rhVEGF), in situ BS-1 lectin staining disclosed enhanced corneal neovascularization in the rhVEGF group compared with controls. These findings were reproduced in mice receiving bone marrow transplanted from transgenic mice constitutively expressing β-galactosidase encoded by lacZ under the transcriptional regulation of an EC-specific gene, tie-2, to establish direct evidence for incorporation of bone marrow–derived EPCs into capillaries and stromal tissue of the corneal neovasculature.
Like fully differentiated ECs,18 EPCs express specific endothelial antigens, including KDR (VEGF receptor-2), CD34, and VE-cadherin.8 9 19 Although KDR and VE-cadherin are generally considered to distinguish EPCs from hematopoietic stem cells,20 21 there exists no epitope of which the expression is restricted exclusively to EPCs versus fully differentiated ECs. There is, however, evidence that EPCs constitute the preponderance of such circulating, bone marrow–derived endothelial lineage cells. First, the present work indicates that the population of circulating EPCs in normal individuals (3 to 5×103/mL) far exceeds the number of differentiated ECs circulating in peripheral blood (2 to 3/mL).22 Second, animal experiments from our own laboratory have suggested that the majority of the cellular population mobilized into the circulation and then incorporated into neovascular foci after VEGF administration is most consistent with bone marrow–derived EPCs.15
These clinical findings call into question certain fundamental concepts regarding the mechanisms by which VEGF promotes blood vessel growth and development in adult organisms. The role of VEGF in postnatal neovascularization has been previously considered synonymous with proliferation and migration of preexisting, fully differentiated ECs resident within parent vessels, ie, sprout formation or angiogenesis.6 12 18 The finding that VEGF augments the number of circulating EPCs in human patients, together with the aforementioned murine experiments,15 implies that its impact on postnatal neovascularization is the combined result of vasculogenesis as well as angiogenesis. The proportional contributions of angiogenesis and vasculogenesis to postnatal neovascularization, including the extent to which each is influenced by VEGF, remain to be clarified.
Finally, these findings have implications for the use of naked DNA in human gene therapy. Earlier studies suggested that the low transfection efficiency associated with the use of naked DNA might make it unsuitable for therapeutic applications in trials of human gene therapy. Subsequent experience in live animal models, however, demonstrated that transfer of genes encoding for secreted proteins, such as VEGF, could yield important biological effects due to the paracrine effects of the secreted gene product.23 The current demonstration that VEGF gene therapy augments the compartment of circulating EPCs constitutes further evidence that gene transfer of naked DNA may indeed be sufficient to modulate the biology of human subjects.
This work was supported in part by NIH Grants HL53354, HL57516, and HL60911 (to J.M.I.) and by a grant from Cologne Fortune Program, Cologne, Germany (to C.K.).
- Received January 14, 2000.
- Accepted May 4, 2000.
- © 2000 American Heart Association, Inc.
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