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Ann Thorac Surg 2000;70:829-834
© 2000 The Society of Thoracic Surgeons
a Department of Vascular Medicine, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA
b Department of Cardiothoracic Surgery, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA
Address reprint requests to Dr Symes, Division of Cardiothoracic Surgery, St. Elizabeth’s Medical Center, 11 Nevins St, MOB 306, Boston, MA 02135
e-mail: jsymes{at}semc.org
Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Methods. The mononuclear cell population was isolated from peripheral venous blood samples of patients with functional class III or IV angina receiving intramyocardial VEGF165 gene transfer. Peripheral blood mononuclear cells were examined by an in vitro EPC culture assay and fluorescent-activated cell sorting. The data were compared with a control group consisting of patients who had undergone off-pump coronary artery bypass grafting without receiving gene transfer.
Results. Coinciding with a rise in VEGF levels, mobilization of EPCs increased significantly over base line for 9 weeks after the treatment (121 ± 14 cells/mm2 versus 36.8 ± 8 cells/mm2, p < 0.0005), followed by a subsequent decrease. Fluorescent-activated cell sorting analysis confirmed culture assay data, with a statistically significant rise in cells expressing vascular endothelial-cadherin, CD51/61 [
vß3], CD62E [E-selectin], CD34, and KDR. The control group failed to show significant mobilization of EPCs.
Conclusions. Mobilization of EPCs with resultant postnatal vasculogenesis, may play a role in revascularizing ischemic myocardium following human gene transfer with VEGF165.
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This approach, referred to as therapeutic angiogenesis, has evolved based on the classic model of angiogenesis, which postulates that it is fully differentiated endothelial cells resident within parent vessels that migrate and then proliferate to bring about new vessel growth in this setting [3, 4]. Recent evidence suggests, however, that postnatal neovascularization may involve an additional pathway. The demonstration that circulating bone-marrow-derived endothelial progenitor cells (EPCs) may home to sites of neovascularization and may differentiate into endothelial cells in situ [5, 6], suggests that vasculogenesis, the embryonic process whereby new vasculature evolves from EPCs or angioblasts, may also participate in the growth and development of new blood vessels in the adult [4, 7, 8]. The mechanism by which EPCs are mobilized within the peripheral circulation has yet to be fully elucidated. Recent data from our laboratory has demonstrated, however, that intraperitoneal administration of VEGF165 protein mobilized EPCs from the bone marrow, resulting in augmented neovascularization in mice [9].
Accordingly we sought to determine whether intramyocardial gene transfer of naked plasmid DNA encoding human VEGF165 may enhance the population of circulating human EPCs in patients with chronic myocardial ischemia.
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Although the initial Phase 1 study did not include a placebo group, a population of 4 patients who underwent off-pump coronary bypass operation served as controls. Two of these patients had manifestations of congestive heart failure, 1 with severe left anterior descending artery disease only, the other with two-vessel disease. Of the other 2 patients, 1 had CCS class III and the other class IV angina.
Plasmid DNA (phVEGF165)
The 13 patients from the study group received an eukaryotic expression vector encoding for the 165-amino-acid isoform of the human VEGF gene transcriptionally regulated by a cytomegalovirus promoter/enhancer. Plasmid DNA was prepared and purified from cultures of phVEGF165-transformed Escherichia coli in the Human Gene Therapy laboratory at St. Elizabeth’s Medical Center using the column method (Qiagen Plasmid Megakit, Valencia, CA) [2].
Myocardial gene transfer
The surgical procedure was performed under general anesthesia utilizing the standard protocol for off-pump coronary bypass operation. The heart was exposed through an 8- to 10-cm, left anterior thoracotomy incision in the fourth or fifth intercostal space. The pericardium was opened and carefully dissected off the epicardial surface of the heart in the apical and the anterolateral region of the left ventricle. A retractor/stabilizer (Cardiothoracic Systems, Cupertino, CA) was inserted to immobilize the epicardial surface over the site of each injection. Continuous transesophageal (TEE) monitoring was used both to assure no change in regional wall motion and, more importantly, to ascertain that the DNA was injected into the myocardium and not into the left ventricular cavity. A total of 250 µg of VEGF165 plasmid DNA was injected in 2-mL aliquots at four separate sites with a 3-mL syringe and 25-gauge needle under direct ultrasound visualization.
The control group of patients underwent off-pump coronary bypass. The procedure was performed using identical anesthesia and monitoring techniques as were used for the gene transfer patients; the retractor stabilizer was also utilized. Transient periods of ischemia averaging about 15 to 20 minutes were induced by occlusion of the coronary vessels in order to perform the anastomoses.
Plasma vascular endothelial growth factor levels
An enzyme-linked immunosorbent assay was used to determine VEGF levels in the plasma in all patients before and at selected time points until 3 months after gene transfer (R & D Systems, Minneapolis, MN). Results were compared with a standard curve of human VEGF with a lower detection limit of 5 pg/mL. Samples were checked by serial dilution and were taken at least in duplicate.
Isolation of peripheral blood mononuclear cells
Blood samples were collected from each patient, before and at selected time points up to 3 months following intramyocardial gene transfer, or off-pump bypass. Approximately 6 mL of each blood sample was collected in a CPT-tube (Becton-Dickinson, San Jose, CA) and total peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation following the manufacturers’ protocol. PBMNCs were harvested and washed twice by Dulbecco’s phosphate-buffered saline (BioWhittaker, Walkersville, MA) supplemented with 5 mmol/L EDTA (DPBS-E). Contaminating red blood cells were hemolysed using ammonium chloride solution (Stem Cell Technologies, Vancouver, Canada). Viable cells were counted in a hemocytometer after Trypan blue 0.4% staining (Gibco, Grand Island, NY).
Endothelial progenitor cell culture assay
The culture system used in our laboratory is a functional index of increased circulating EPCs and has been described elsewhere [9]. Immediately following isolation, PBMNCs from 500 µL peripheral blood (typically 7 x 105 to 1.4 x 106 cells) were plated on four-well glass slides coated with human fibronectin (Sigma, St. Louis, MO) and maintained in endothelial cell basal medium-2 (EBM-2) (Clonetics, San Diego, CA). The media was supplemented with EGM-2 MV Single Quots (Clonetics) including 5% fetal bovine serum, 0.5 mL human epidermal growth factor, 0.5 mL human vascular endothelial growth factor-1, 2 mL human fibroblast growth factor-2, 0.5 mL insulin-like growth factor-1, and 0.5 mL ascorbic acid. After 4 days in culture, nonadherent cells were removed by thorough washing with PBS and adherent cells underwent cytochemical analysis. Fluorescent chemical staining was used to detect the binding of fluorescein isothiocyanate (FITC)-labeled Ulex europaeus (Sigma), and the uptake of acetylated low-density lipoprotein labeled with DiI (acLDL-DiI, Biomedical Technologies, Stoughton, MA) at a concentration of 10 µg/mL. After the staining procedures, the samples were viewed with an inverted fluorescence microscope (Nikon, Tokyo, Japan). Dual fluorescent staining positive for both FITC-labeled Ulex and acLDL-DiI (double-positive cells) were identified as differentiating EPCs. Two independent investigators evaluated the number of EPCs per well by counting 20 randomly selected fields. The mean value of this calculation was then expressed as cells/mm2. Human umbilical vein endothelial cells were prepared as described previously [10] and used between passages 4 and 6 serving as positive controls. Neonatal human dermal fibroblasts (Clonetics) served as negative controls.
Flow cytometry analysis
Samples from gene transfer patients and individuals undergoing bypass operation were collected at base line and at 1, 2, 4, and 9 weeks after the treatment. Approximately 5 x 106 cells were immediately fixed in 1% paraformaldehyde and stored at 4°C in PBS in preparation for fluorescent-activated cell sorting (FACS). A total of 2 x 105 to 3 x 105 cells in PBS with 10% fetal bovine serum were incubated for 30 minutes at 4°C with monoclonal antibodies prepared against human KDR (Sigma), recognizing the extracellular domain, and against human vascular endothelial (VE)-cadherin (clone BV 6, mouse IgG2a, gift from E. Dejana). Further antibodies tested included the biotinylated CD62E (E-selectin), the FITC-conjugated hCD51/61 (vß3) (Pharmingen, San Diego, CA), and the phycoerythrin-conjugated hCD31 and hCD34 (Becton Dickinson). For analysis of KDR and VE-cadherin, cells were further incubated with a biotinylated anti-mouse IgG (H+L) antibody (Vector Laboratories, Burlingame, CA), and with FITC-conjugated streptavidin, both for 30 additional minutes. Quantitative FACS analysis was performed on a FACStar flow cytometer (Becton Dickinson). Histograms of cell number versus logarithmic fluorescence intensity were recorded for 10,000 to 20,000 cells per sample.
Statistical analysis
All results are expressed as mean ± standard error (±SEM). Statistical significance was evaluated using unpaired Student’s t test for comparisons between two means. A value of p less than 0.05 was interpreted to denote statistical significance.
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vß3]: 3.1 x 103 versus 2 x 104, p < 0.02 and CD62E [E-selectin]: 7.2 x 104 versus 2 x 105, p = 0.05). The control group by comparison failed to show significant mobilization of EPCs (VE-cadherin: 1.7 x 103 versus 3.3 x 103, p = 0.16; KDR: 3.6 x 103 versus 6 x 103, p = 0.23; CD34 3.1 x 104 versus 5.1 x104, p = 0.32; CD51/61 [vß3]: 1 x 103 versus 3.3 x 103, p = 0.21 and CD62E [E-selectin]: 1.6 x 104 versus 4.6 x 104, p = 0.28). Posttreatment values in the VEGF-treated group were also significantly different compared with the control group (for VE-cadherin, p < 0.05; for KDR, p < 0.02; for CD34, p < 0.05; for CD51/61 [vß3], p < 0.02; and for CD62E [E-selectin], p < 0.05).
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In the present study we demonstrated, in addition, a modulation of EPC kinetics following intramyocardial VEGF gene transfer. Coinciding with the rise in plasma levels of VEGF, the number of EPCs in the peripheral blood increased significantly over base line as early as 1 week after gene transfer, and remained elevated for up to 9 weeks. By contrast, the EPC kinetics in the off-pump CABG group remained unchanged. These patients were similar clinically in terms of their angina class and had undergone transient episodes of myocardial ischemia and reperfusion during the procedure, both of which might be expected to upregulate endogenous VEGF and other angiogenic cytokines. The absence of any long-term mobilization of EPC postoperatively in these patients suggests that the longer duration and increased expression of VEGF protein associated with gene transfer is required to induce vasculogenesis in this setting.
Both fully differentiated endothelial cells (ECs) and EPCs share the common surface antigens KDR, CD 34, Tie-2, and VE-cadherin [11–13]. Unfortunately, no epitope exists whose expression is restricted exclusively to EPCs versus fully differentiated ECs. Nonetheless, there is evidence that EPCs constitute the preponderance of such circulating, bone marrow-derived endothelial lineage cells. First, the number of differentiated ECs in the peripheral blood (2 to 3 per milliliter) [14, 15] appears to be considerably lower than the population of circulating EPCs in normal individuals (3 x 103 to 4 x 103 per milliliter) (Christoph Kalka, unpublished data). Second, our own murine experiments suggest that most of the cellular population mobilized into the circulation by VEGF and homing to foci of neovascularization consists of bone marrow-derived EPCs [9].
Limitations in our ability to analyze the origin and fate of the mobilized population of circulating EPCs in human subjects requires making inference from animal experiments. Preclinical studies in mice demonstrated that daily intraperitoneal injections of recombinant human VEGF165 (rhVEGF) resulted in an increased number of circulating EPCs, an effect that was abrogated by simultaneous application of a neutralizing antibody to rhVEGF [9]. The same treatment showed enhanced corneal neovascularization in the rhVEGF group compared with controls. Transplantation models using mice transplanted with bone marrow from transgenic mice constitutively expressing ß-galactosidase encoded by lacZ under the transcriptional regulation of an EC-specific gene, tie-2, established that bone marrow-derived EPCs incorporated into capillaries and stromal tissue of the corneal neovasculature [7]. These experimental data suggest therefore that EPCs mobilized by VEGF do indeed participate in the process of neovascularization of ischemic tissue.
The presented clinical findings call into question certain fundamental concepts regarding blood vessel growth and development in adult organisms. Previously, postnatal neovascularization had been considered synonymous with proliferation and migration of preexisting, fully differentiated ECs resident within parent vessels, consistent with the classic paradigm of angiogenesis [16]. The finding that circulating EPCs may home to sites of neovascularization [6, 7, 17] and differentiate into ECs in situ is consistent with vasculogenesis [4], a critical paradigm for establishment of the primordial vascular network in the embryo. Although the proportional contributions of angiogenesis and vasculogenesis to postnatal neovascularization remain to be clarified, our findings together with recent reports from other investigators [6, 15], suggest that growth and development of new blood vessels in the adult is not restricted to angiogenesis but encompass both embryonic pathways.
Additionally, animal studies and our preliminary clinical results in patients with critical limb ischemia suggest that endogenous angiogenesis is impaired as a result of endothelial dysfunction in the presence of advanced age as well as diabetes and hypercholesterolemia [18–20]. This finding, however, does not appear to preclude a favorable response to exogenous VEGF administration perhaps implying that VEGF-induced EPC mobilization may provide an enlarged pool of endothelial cells capable of participating in the process of neovascularization.
In conclusion, human gene transfer with VEGF appears to mediate neovascularization by effecting both the angiogenic and vasculogenic pathways. Further investigations aimed at deciphering the mechanisms underlying EPC kinetics and their biological effects will widen our understanding of this process with the potential to expand the possibilities for therapeutic neovascularization in patients with otherwise unrevascularizable coronary artery disease.
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S. Shintani, T. Murohara, H. Ikeda, T. Ueno, T. Honma, A. Katoh, K.-i. Sasaki, T. Shimada, Y. Oike, and T. Imaizumi Mobilization of Endothelial Progenitor Cells in Patients With Acute Myocardial Infarction Circulation, June 12, 2001; 103(23): 2776 - 2779. [Abstract] [Full Text] [PDF]
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H. Iwaguro, J.-i. Yamaguchi, C. Kalka, S. Murasawa, H. Masuda, S.-i. Hayashi, M. Silver, T. Li, J. M. Isner, and T. Asahara Endothelial Progenitor Cell Vascular Endothelial Growth Factor Gene Transfer for Vascular Regeneration Circulation, February 12, 2002; 105(6): 732 - 738. [Abstract] [Full Text] [PDF]
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) compared with the number of EPCs (
), as determined by the in vitro culture assay, at base line, and selected time points after phVEGF165 gene transfer. Vascular endothelial growth factor plasma increased 2.9-fold over base line at the observation time point of 1 week (p < 0.02) and remained 1.5-fold elevated up to 4 weeks after gene transfer. The measurements of EPCs in vitro revealed a coinciding 3.5-fold elevation over base line as early as 1 week after therapy (p < 0.02) with a sustained increase up to 4 weeks.
coronary bypass surgery, 
VEGF gene transfer) as determined by the EPC culture assay, and VEGF plasma levels (
coronary bypass surgery, 