This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosengart, T. K. Articles by Crystal, R. G. Search for Related Content PubMed PubMed Citation Articles by Rosengart, T. K. Articles by Crystal, R. G.

(Circulation. 1999;100:468-474.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

Angiogenesis Gene Therapy

Phase I Assessment of Direct Intramyocardial Administration of an Adenovirus Vector Expressing VEGF121 cDNA to Individuals With Clinically Significant Severe Coronary Artery Disease

Todd K. Rosengart, MD; Leonard Y. Lee, MD; Shailen R. Patel, MD; Timothy A. Sanborn, MD; Manish Parikh, MD; Geoffrey W. Bergman, MBBS; Rory Hachamovitch, MD; Massimiliano Szulc, PhD; Paul D. Kligfield, MD; Peter M. Okin, MD; Rebecca T. Hahn, MD; Richard B. Devereux, MD; Martin R. Post, MD; Neil R. Hackett, PhD; Taliba Foster, MS; Tina M. Grasso, BS; Martin L. Lesser, PhD; O. Wayne Isom, MD; Ronald G. Crystal, MD

From the Department of Cardiothoracic Surgery (T.K.R., L.Y.L., S.R.P., O.W.I.), Division of Pulmonary and Critical Care Medicine (L.Y.L., S.R.P., N.R.H., T.F., R.G.C.), Division of Cardiology (T.A.S., M.P., G.W.B., R.H., M.S., P.D.K., P.M.O., R.T.H., R.B.D., M.R.P.), Weill Medical College of Cornell University–New York Presbyterian Hospital, New York, NY; GenVec, Inc (T.M.G.), Rockville, Md; and Division of Biostatistics (M.L.L.), North Shore University Hospital, Manhasset, NY.

Correspondence to R.G. Crystal, MD, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, NY 10021. E-mail geneticmedicine{at}mail.med.cornell.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Therapeutic angiogenesis, a new experimental strategy for the treatment of vascular insufficiency, uses the administration of mediators known to induce vascular development in embryogenesis to induce neovascularization of ischemic adult tissues. This report summarizes a phase I clinical experience with a gene-therapy strategy that used an E1^-E3^- adenovirus (Ad) gene-transfer vector expressing human vascular endothelial growth factor (VEGF) 121 cDNA (Ad_GVVEGF121.10) to induce therapeutic angiogenesis in the myocardium of individuals with clinically significant coronary artery disease.

Methods and Results—Ad_GVVEGF121.10 was administered to 21 individuals by direct myocardial injection into an area of reversible ischemia either as an adjunct to conventional coronary artery bypass grafting (group A, n=15) or as sole therapy via a minithoracotomy (group B, n=6). There was no evidence of systemic or cardiac-related adverse events related to vector administration. In both groups, coronary angiography and stress sestamibi scan assessment of wall motion 30 days after therapy suggested improvement in the area of vector administration. All patients reported improvement in angina class after therapy. In group B, in which gene transfer was the only therapy, treadmill exercise assessment suggested improvement in most individuals.

Conclusions—The data are consistent with the concept that direct myocardial administration of Ad_GVVEGF121.10 to individuals with clinically significant coronary artery disease appears to be well tolerated, and initiation of phase II evaluation of this therapy is warranted.

Key Words: angiogenesis • gene therapy • genetics • coronary disease • ischemia

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Anew experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by use of "angiogens," mediators that induce the formation of blood vessels.^{1 2} This approach is based on the knowledge that in the adult heart, the genes coding for angiogens and their receptors are expressed in low levels, apparently insufficient in most individuals to provide robust formation of collaterals in response to chronic ischemia.^{3 4}

Vascular endothelial growth factor (VEGF), a protein coded by a 7-exon gene localized on chromosome 6, serves as a major angiogen in normal cardiac development.⁵ The VEGF gene is normally spliced into 4 different forms; of these, VEGF121 (containing 121 amino acids) and VEGF165 (165 amino acids) appear to be the most important. The VEGF proteins function by interacting with specific receptors on endothelial cells, which initiates a cascade of events culminating in endothelial cell migration, proliferation, aggregation into tubelike structures, and networking of the arterial and venous systems.^{2 5 6 7 8}

Gene transfer represents one approach to delivering an angiogen to the heart in which the cDNA coding for VEGF is delivered to the myocardium, with the myocardial cells used to secrete the VEGF.^{8 9 10} Studies in experimental animals have shown that replication-deficient, recombinant adenovirus (Ad) gene-transfer vectors are advantageous for delivery of angiogens like VEGF in that Ad vectors provide a high transfection efficiency, remain highly localized, and express VEGF for a period of 1 to 2 weeks, which is sufficient to induce collateral vessels to relieve the ischemia but not long enough to evoke abnormal angiogenesis.^{8 9 10 11 12 13}

Based on experimental animal models demonstrating the development of new blood vessels after in vivo administration of an Ad vector expressing human VEGF121 cDNA, including anatomic and functional correction of ischemia in a pig model of coronary obstruction,⁸ the present study was directed toward evaluation of the administration of an E1^-E3^- Ad vector (Ad_GVVEGF121.10) expressing the 121-amino-acid form of human VEGF to individuals with clinically significant coronary artery disease. The Ad_GVVEGF121.10 vector was administered directly to an ischemic area of the myocardium as an adjunct to conventional CABG surgery in a region that could not be bypassed (group A) or through a minithoracotomy as sole therapy (group B).

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Ad_GVVEGF121.10
The Ad gene-transfer vector Ad_GVVEGF121.10 (GenVec, Inc) is based on the genome of the Ad5 serotype, with deletions in the E1 and E3 regions.⁸ The expression cassette is in the E1 region and contains (right to left) the cytomegalovirus early/immediate enhancer/promoter, an artifical splice sequence, human VEGF121 cDNA, and the SV40 polyA/stop signal. Ad_GVVEGF121.10 was propagated in 293 cells, purified by CsCl density gradients, dialyzed, and stored at -70°C.^{8 14} The vector met all safety criteria established by the Food and Drug Administration Bureau of Biologics (FDA BB) for clinical grade Ad vector preparations (FDA BB-IND 7381), including no detectable endotoxin or infectious agents and 1 replication-competent Ad for the total dose to be delivered.¹⁵ The vector was titered in plaque-forming units (pfu)¹⁴ and characterized as to particle units (pu) with the absorbance at 260 nm and the extinction coefficient for Ad (9.09x10^-12 mL · particles^-1 · cm^-1).¹⁶ Just before use, the vector was thawed, diluted in a 3% sucrose solution, drawn up as 100 µL in 1-mL-insulin syringes with a 27-gauge needle (Becton Dickinson), and transported to the operating room.

Study Design
The study, which was approved by the local Institutional Review Board and the NIH DNA Recombinant Advisory Committee, was similar for groups A and B and included men and women aged 18 to 85 years with demonstrable reversible left ventricular ischemia as assessed by dobutamine stress echocardiography, rest and stress ^99mTc-sestamibi nuclear medicine studies, and exercise tolerance testing. Twenty-four-hour Holter monitoring was used to exclude individuals with life-threatening arrhythmias. Other organ-specific inclusion criteria included room air PO₂ >60 mm Hg, PCO₂ <50 mm Hg, FEV₁ >1.2 L, hematocrit >30%, white blood cell count <10 000, serum urea nitrogen <40 U/L, and creatinine <2.5 g/dL. Group A (adjunct) had a requirement for an ejection fraction of 25% and 1 bypassable vessel, with the vector to be administered in a viable, ischemic region not amenable to bypass grafting. Group B (sole therapy) had a requirement of an ejection fraction 30% and included patients in whom CABG could not be performed due to lack of suitable bypass graft targets.

Ad_GVVEGF121.10 was administered by direct myocardial injection to both group A and B patients in a myocardial territory, irrespective of size, that demonstrated reversible ischemia by ^99mTc-sestamibi perfusion scan with or without adenosine stress. The injections (100 µL/injection; 10 sites/patient; each site 1 to 1.5 cm apart) were administered to a region that extended from normal (bypassed) myocardium into the ischemic (nonbypassed) territory for collateral vessels that would bridge the myocardial territory from a patent inflow vessel to an ischemic territory and in which no continuous patent epicardial vessel was observed by angiography. For group A, once the CABG procedure was completed through a standard median sternotomy, the patient was rewarmed to 36°C, and the vector was administered to the myocardium while the patient was supported by partial bypass. Five dose groups were evaluated (n=3 patients per dose group), with total doses as follows: 4x10⁸, 4x10^8.5, 4x10⁹, 4x10^9.5, and 4x10¹⁰ pu. For group B (sole-therapy group, n=6), a small (4 to 5 cm) thoracotomy was used to expose the region of the myocardium chosen for vector administration. The vector (total dose 4x10⁹ pu/patient) was then injected by direct visualization in the beating heart into a region of reversible ischemia.

General Safety Parameters
Blood parameters, including aspartate aminotransferase, alanine aminotransferase, bilirubin (total, direct, and indirect), alkaline phosphatase, albumin, white blood count, hemoglobin, hematocrit, platelet count, electrolytes, creatinine, serum urea nitrogen, lactate dehydrogenase, and creatine kinase (CK; CK-MB if the total CK was abnormal), were measured through the perioperative period and at days 14, 21, and 30 postoperatively.

Anti-Ad5 neutralizing antibody titers were assayed as previously described.¹⁷ Plasma levels of VEGF were determined by standard ELISA; the assay detects all forms of human VEGF.^{8 11} The samples were obtained in citrate tubes (Vacutainer L10278-00 2.7 mL; Becton Dickinson) to avoid contamination with platelet-derived VEGF.¹⁸ Nose, throat, urine, and blood samples (before therapy and on days 2, 4, and 7) were evaluated for both E1^- Ad vector and wild-type Ad.¹⁵

Cardiac-Specific Parameters
The degree of angina (on a scale of 1 to 4) was assessed preoperatively and 30 days after surgery by use of a questionnaire describing the Canadian Cardiovascular Society classification.¹⁹ Serial ECG was used to assess myocardial ischemia, infarction, or arrhythmia. In the adjunct group, 24-hour Holter monitoring was performed before therapy and at 7 days after therapy.²⁰

Biplanar contrast angiography was performed preoperatively within 2 months of the surgical procedure and at day 30 after therapy. The angiograms were reviewed by 3 interventional cardiologists blinded to treatment group and evaluated in the area of vector administration on the basis of Rentrop score (0 indicates no filling of collateral; 1, partial filling of branches of epicardial vessel; 2, partial filling of epicardial vessel; and 3, complete filling of epicardial vessel)²¹ and collateral score (number of distinct collateral vessels contributing to the filling of an epicardial vessel in the region of vector administration).⁸ All studies were read in random sequence, and samples from before and after the study were randomly presented to observers.

A 2-day combined rest-stress ^99mTc-sestamibi study to assess myocardial viability was performed preoperatively within 2 weeks of the surgery and at 1 month after surgery. One hour after intravenous administration of ^99mTc-sestamibi (25 to 30 mCi), ECG-gated single-photon emission computerized tomography (SPECT) imaging was performed with or without pharmacological stress with adenosine (140 µg · kg^-1 · min^-1 IV over 6 minutes). Semiquantitative analyses of perfusion were assessed by use of a 20-segment analysis (18 short axis and 2 long axis) in a blinded fashion by 2 nuclear cardiologists and scored in the region of vector administration on a scale of 0 to 4+, where 0 indicates no perfusion, 1 is severe hypoperfusion, 2 is moderate hypoperfusion, 3 is mild hypoperfusion, and 4 is normal perfusion. Using CEqual software (ADAC), we generated "bull's-eye" images for rest scans, stress scans, and their differences ("reversibility" of stress-induced ischemia) quantified as a percentage of the entire myocardium compared with a sex-matched normal database.²²

Serial 2D echocardiography was used to determine the presence of pericardial effusion at baseline (within 2 weeks of operative procedure) and on days 2, 4, 7, 14, 21, and 30 postoperatively by a 0 to 3+ scale (0 indicates no effusion, 1 is mild effusion, 2 is moderate effusion, and 3 is large effusion). Regional wall motion at rest was assessed in the region of vector administration at baseline and on day 30 by an observer blinded to treatment groups, using a scale from 0 to 4+, where 0 indicates dyskinesis/akinesis, 1 is severe hypokinesis, 2 is moderate hypokinesis, 3 is mild hypokinesis, and 4 is normal.

Exercise tolerance testing was performed preoperatively and at day 30 according to a modified Bruce protocol.²³ Peak heart rate, peak heart ratexpeak systolic blood pressure, and ST-segment/heart rate (ST/HR) slope (from peak exercise regression of ST depression expressed as a positive value referenced to heart rate) were determined.²³

Statistical Analyses
Given that this is a phase I clinical trial, the number of patients at each dose in group A (n=3) and the total number of patients in group B (n=6) are too few to provide sufficient statistical power to discriminate within the variability of the various parameters that were assessed. Therefore, lack of statistical significance may not necessarily be interpreted as "no difference." The results are presented without formal error estimates and in the context of trends suggested by the data.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Patient Demographics
The Ad_GVVEGF121.10 vector was administered to the myocardium as an adjunct to CABG (group A) in 15 patients (Table 1). The patients who were undergoing sole gene therapy (group B) were of similar age and had similar risk factors to those in group A but had a trend to a greater degree of cardiac disease, including an average lower ejection fraction and a higher proportion having undergone a prior CABG or angioplasty procedure.

View this table: [in this window] [in a new window]   Table 1. Demographics and Intraoperative and Postoperative Characteristics of the Study Population

Vector Administration
In group A, the region of injection was in the distribution of the left anterior descending (LAD) or circumflex coronary artery in the majority of patients, with the remainder in other sites (Table 1). One individual in group A (A10, vector dose 4x10^9.5 pu) underwent cardiopulmonary bypass, but no bypass graft was placed because of the severity of distal disease as assessed intraoperatively. In group B, 5 of 6 individuals were injected in either the LAD or circumflex territories. The average time required for injection was 3.9 minutes in group A and 6.0 minutes in group B; the time was longer in group B because of technical constraints imposed by the minimally invasive approach. Minimal extravasation of injectant was noted in both groups. Occasional premature ventricular beats were observed with insertion of the needle into the myocardium but were self-limited in all cases.

General Outcome
In group A, there were 2 perioperative (within 40 days of operative procedure) deaths. One, on postoperative day 40 in a 61-year-old male (A5, vector dose 4x10^8.5 pu) who was undergoing a third CABG operation, was related to a large anterior wall myocardial infarction secondary to occlusion of a graft to the LAD artery. Autopsy revealed a bacterial pneumonia and lung abscess; there were no abnormalities in the myocardial territory (posterior descending coronary artery) treated with the vector. The second death occurred on postoperative day 5 in an 85-year-old female (A15, vector dose 4x10¹⁰ pu) secondary to complications associated with an atheroembolic event in the ileocolic artery distribution. There was 1 additional sudden death of unknown cause (patient A14, dose 4x10¹⁰ pu, day 145 after therapy) in group A (mean±SD follow-up 286±76 days; range 175 to 414 days).

In group B, patients were extubated in the operating room, observed in the recovery room until awake, and transferred to the routine care floor until discharge. There were no perioperative or late deaths (mean±SD follow-up 170±17 days; range 149 to 196 days).

General Safety Parameters
There was no evidence of a dose-related trend toward abnormalities in any blood parameters in group A and no differences in any blood parameters for group B at day 3, 7, or 30 compared with before therapy. Plasma VEGF levels were evaluated over a 30-day period after therapy in the individuals who received 4x10^9.5 and 4x10¹⁰ pu in the adjunct group and in all patients in the sole-therapy group. There were no trends to increases above baseline levels except at day 3, when the average value was 158 pg/mL. There was no evidence of acute or sustained hypotension or hemodynamic compromise associated with sole therapy (group B). In both groups, serum anti-Ad5 neutralizing antibody levels were increased in all individuals, although more so in patients with higher pretherapy anti-Ad5 neutralizing antibodies (not shown). No shedding of vector or wild-type Ad was detected in any sample from any site in any patient (group A total 234 samples; group B total 71 samples).

Cardiac-Related Parameters
In group A, there was no dose-related trend of an increase in CK related to vector administration (Table 2). In group B, there was no increase in CK after therapy (Table 2). In either group, daily ECG during hospitalization and at 14 and 30 days showed no new ST changes or Q waves (Table 3). In group A, 24-hour Holter monitoring performed before therapy and at day 7 demonstrated no average increase in supraventricular or ventricular arrhythmias after therapy. Serial echocardiographic studies in both groups demonstrated no evidence of significant (2) pericardial effusions. In both groups, resting echocardiographic assessment at day 30 compared with before therapy showed no regional wall motion abnormalities in the territory where the vector was administered.

View this table: [in this window] [in a new window]   Table 2. CK Values of Patients Receiving AdGV VEGF121.10 (109 pu) as Sole Therapy

View this table: [in this window] [in a new window]   Table 3. Comparison of ECG at Baseline and Postvector Day 30 for Patients Receiving AdGV VEGF121.10

Assessment of angina class in group A showed improvement in all individuals evaluated, but this cannot be attributed specifically to the Ad_GVVEGF121.10 therapy because of the effects of bypass. However, in all 6 of the individuals in group B, there was a decrease in angina classification at day 30 compared with before therapy (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Canadian angina classifications before and 30 days after intramyocardial delivery of AdGVVEGF121.10 (4x109 pu) as sole therapy. Each before-and-after pair represents 1 individual. Only a single dose was used.

Coronary angiograms obtained 30 days after vector administration demonstrated no hemangiomas or other pathological vascular structures. In group A, a majority of the blinded observations demonstrated an improvement in Rentrop scores in the area of vector administration after therapy compared with before therapy (Figure 2A). The collateral scores in group A demonstrated a similar trend (Figure 2B). Likewise, in group B, in which no CABG was performed that might provide a watershed effect in the area treated with the vector, a majority of the Rentrop and collateral score observations showed a similar trend of improvement after therapy compared with before therapy (Figure 2C).

View larger version (19K): [in this window] [in a new window]   Figure 2. Semiquantitative, blinded assessment of coronary angiograms before and 30 days after intramyocardial administration of AdGVVEGF121.10. A, Rentrop scores, adjunct to CABG. B, Collateral scores, adjunct to CABG. C, Rentrop and collateral scores, sole therapy/minithoracotomy. For all panels, a positive value (score at 30 days after therapy minus before therapy) indicates improvement at 30 days compared with before therapy. , Observer 1; , observer 2; , observer 3.

For group A assessed as a single cohort, semiquantitative analysis of the ^99mTc-sestamibi images in the area of vector administration showed no changes in relative blood flow at 30 days after vector administration compared with pretherapy at rest or after adenosine-induced stress. Likewise, for group B evaluated as a cohort, analysis of the sestamibi images demonstrated no differences in relative blood flow in the area of vector administration at rest or after adenosine-induced stress. Interestingly, analysis of the sestamibi images for wall motion at stress in the region of vector administration showed an improvement at 30 days after vector administration in the majority of patients, both in group A (66% [8/12] improved) and in group B (66% [4/6] improved). For group B, in which bull's-eye analyses of the sestamibi scans could be performed with assessment of vector administration as the only variable, 4 of 6 individuals showed an improvement 30 days after therapy in the proportion of myocardium that showed reversibility (reversible stress-induced ischemia) before therapy (70±31%) versus 30 days after therapy (54±36%).

Assessment of treadmill exercise in group A showed no differences (30 days after vector compared with before therapy) in exercise duration, heart ratexblood pressure, or ST/HR slope. For group B, in which vector administration was the only therapy, assessment of treadmill exercise showed an improvement in exercise duration in 50% (3/6), in peak heart ratexblood pressure in 50% (3/6), and in ST/HR slope (ie, lower values) in 75% (3/4; data not available in 2 secondary to right bundle-branch block precluding analysis; Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 3. Treadmill exercise before and 30 days after intramyocardial administration of AdGVVEGF121.10 in group B (sole therapy). Shown is percent improvement for each patient at 30 days after vector compared with baseline [(30-days posttherapy- pretherapy)x100/pretherapy] for duration of exercise, blood pressurexheart rate (HR x BP), and ST-segment depression/heart rate (ST/HR). *ST/HR slope plotted as negative of percent change from baseline; ie, a positive score reflects a reduction in test value to a less abnormal result.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The development of strategies to deliver angiogens to revascularize the ischemic myocardium without the need for mechanical manipulation of atherosclerotic vessels is potentially of profound importance in the treatment of coronary artery disease. The present study demonstrates that it is feasible to safely use an adenovirus gene-transfer vector to deliver the coding sequence of the 121-amino-acid form of the human VEGF angiogen to the myocardium of individuals with clinically significant coronary artery disease. Based on the knowledge that VEGF plays a critical role in embryonic cardiac angiogenesis²⁴ and on preclinical studies that demonstrate that the Ad_GVVEGF121.10 gene-transfer vector will induce functional angiogenesis in the ischemic myocardium of experimental animals,⁸ the results of the present study provide encouragement that this strategy may be useful in revascularizing the ischemic heart in humans.

Cardiac-Specific Parameters
There was no evidence of either myocardial inflammation or necrosis, as was shown by the lack of dose-related increases in CK, arrhythmias or ST/T wave changes assessed by Holter and ECG monitoring, and deterioration of global or segmental function in the area of vector administration as assessed by echocardiography or ^99mTc-sestamibi. There was no evidence of excess deranged angiogenesis, as evidenced by no hemangiomas or otherwise deranged vasculature in the coronary angiograms and no evidence of myocardial edema or pericranial effusions. These observations are consistent with the assessment of the safety of administration of human VEGF165 cDNA plasmids to the human myocardium, either by intracoronary administration²⁵ or by direct myocardial administration.⁹

The present study is limited by the number of cases being too small to provide sufficient power to discriminate within the variability of the various methods used to assess cardiac function. Furthermore, although the vector was delivered in group A to a myocardial territory that could not be bypassed, the vector was administered in conjunction with a conventional CABG procedure, and thus it is impossible to exclude the possibility of CABG-related watershed perfusion affecting the region of vector administration. Despite these constraints, the trends of several of the efficacy-related parameters assessed 30 days after therapy are encouraging. First, all patients had improvement in their angina classification. Although this can be ascribed to the CABG procedure in group A, there was a similar trend to improvement in the sole-therapy group. Second, in the majority of individuals in both groups A and B, angiographic studies showed increased coronary artery filling and/or number of collaterals in the region of vector administration. Third, the majority of individuals in groups A and B had improvement in ventricular wall motion with stress as assessed by ^99mTc-sestamibi scans. Finally, the majority of individuals in the sole-therapy group had evidence of decreased stress-induced reversible ischemia on sestamibi perfusion scans, as well as improvements in treadmill exercise parameters. The observed decrease in reversible ischemia could theoretically be caused by infarction of this territory, but this is unlikely, because there were no corresponding infarction-related changes in CK, ECG or echocardiography.

Systemic Parameters
Assessment of blood and urine parameters suggested no systemic abnormalities related to the vector, consistent with the general clinical experience with E1^- Ad gene transfer to humans.^{26 27} Importantly, there was no evidence of liver function abnormalities as a function of vector dose; this is important because the liver is a major site of Ad vector–induced inflammation at high doses in some studies in experimental animals.^{28 29} One explanation for this lack of systemic toxicity in the human studies is that the vector preparations used in clinical studies are highly purified (<1 replication-competent Ad [RCA] per total dose), in contrast to laboratory-grade vectors, which are often contaminated with RCAs.¹⁵

Myocardial administration of Ad_GVVEGF121.10 induced an increase in anti-Ad neutralizing antibodies in most of the study population. This was more pronounced in individuals with detectable serum anti-Ad neutralizing antibodies before therapy, consistent with the vector inducing a memory immune response against subgroup C Ad.^{15 29 30} Despite this, there was no evidence of systemic immune-related toxicity in any patient, including no immediate anaphylactic-type reactions, vasculitis, or renal damage.

Finally, it is known that systemic administration of the VEGF protein at high doses results in systemic hypotension in experimental animals and humans.³¹ However, the present study demonstrated no large increases in VEGF levels in the systemic circulation after myocardial administration of Ad_GVVEGF121.10 and no hypotension attributable to the vector, consistent with experimental animal studies using Ad_GVVEGF121.10.⁸

Future Role of Angiogenic Gene Therapy
The ability to biologically revascularize tissues, if proven to be safe and efficacious in large, controlled trials, will be an invaluable treatment for patients with diffuse disease not amenable to conventional CABG or PTCA and may be useful as initial therapy in some individuals in place of routine CABG or PTCA therapy. In the present study, we used an Ad vector to deliver the VEGF121 cDNA. As an alternative, Losordo et al⁹ used myocardial administration of a VEGF165 plasmid as sole therapy for myocardial ischemia and demonstrated a safety profile and trends in efficacy parameters similar to our study. If one assumes that the neovasculature induced by angiogenic therapy is persistent and physiologically relevant, the small-caliber vessels generated by this therapy may furthermore be relatively spared from the effects of atherosclerosis, which primarily affects larger vessels. Finally, given the decreased survival overall and decreased angina-free survival noted in patients in whom incomplete revascularization is accomplished, the advantages of providing "complete" revascularization in patients undergoing standard CABG or PTCA may also prove be a significant benefit of this new therapy.

	Acknowledgments

 
These studies were supported in part by NIH R01 HL-57318 and M01RR00047; Jeffry M. and Barbara Picower Foundation, Palm Beach, Fla; Will Rogers Memorial Fund, Los Angeles, Calif; GenVec, Inc, Rockville, Md; and Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Mich. We would like to thank Stephanie Rempel and Sharon Bauer for help with the study and N Mohamed for help in preparation of this manuscript.

	Footnotes

 
Dr Rosengart is a consultant to GenVec, Inc, a privately held biotechnology company that is engaged in the business of designing and manufacturing vectors. Dr Crystal has equity in and is a consultant to GenVec, Inc.

Received February 8, 1999; revision received May 9, 1999; accepted May 13, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Rosengart TK, Patel SR, Crystal RG. Therapeutic angiogenesis: protein and gene therapy delivery strategies. J Cardiovasc Res. 1999;6:29–40.
   
2. Simons M, Ware JA. Food for starving hearts. Nat Med. 1996;2:519–520.[Medline] [Order article via Infotrieve]
   
3. Banai S, Shweiki D, Pinson A, Chandra M, Lazarovici G, Keshet E. Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Cardiovasc Res. 1994;28:1176–1179.[Abstract/Free Full Text]
   
4. Li J, Brown LF, Hibberd MG, Grossman JD, Morgan JP, Simons M. VEGF, flk-1, and flt-1 expression in a rat myocardial infarction model of angiogenesis. Am J Physiol. 1996;270:H1803–H1811.[Abstract/Free Full Text]
   
5. Ferrara N, Houck KA, Jakeman LB, Winer J, Leung DW. The vascular endothelial growth factor family of polypeptides. J Cell Biochem. 1991;47:211–218.[Medline] [Order article via Infotrieve]
   
6. Folkman J, Haudenschild C. Angiogenesis in vitro. Nature. 1980;288:551–556.[Medline] [Order article via Infotrieve]
   
7. Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem. 1996;271:603–606.[Free Full Text]
   
8. Mack CA, Patel SR, Schwarz EA, Zanzonico P, Hahn RT, Ilercil A, Devereux RB, Goldsmith SJ, Christian TF, Sanborn TA, Kovesdi I, Hackett N, Isom OW, Crystal RG, Rosengart TK. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg. 1998;115:168–176; discussion 176–177.[Abstract/Free Full Text]
   
9. Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation. 1998;98:2800–2804.[Abstract/Free Full Text]
   
10. Magovern CJ, Mack CA, Zhang J, Hahn RT, Ko W, Isom OW, Crystal RG, Rosengart TK. Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector. Ann Thorac Surg. 1996;62:425–433; discussion 433–434.[Abstract/Free Full Text]
    
11. Mack CA, Magovern CJ, Budenbender KT, Patel SR, Schwarz EA, Zanzonico P, Ferris B, Sanborn T, Isom P, Isom OW, Crystal RG, Rosengart TK. Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion. J Vasc Surg. 1998;27:699–709.[Medline] [Order article via Infotrieve]
    
12. Muhlhauser J, Merrill MJ, Pili R, Maeda H, Bacic M, Bewig B, Passaniti A, Edwards NA, Crystal RG, Capogrossi MC. VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res. 1995;77:1077–1086.[Abstract/Free Full Text]
    
13. Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol Cell. 1998;2:549–558.[Medline] [Order article via Infotrieve]
    
14. Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L, Perricaudet M, Guggino WB, Pavirani A, Lecocq J-P, Crystal RG. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992;68:143–155.[Medline] [Order article via Infotrieve]
    
15. Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet. 1994;8:42–51.[Medline] [Order article via Infotrieve]
    
16. Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol. 1996;70:7498–7509.[Abstract]
    
17. Mack CA, Song WR, Carpenter H, Wickham TJ, Kovesdi I, Harvey BG, Magovern CJ, Isom OW, Rosengart T, Falck-Pedersen E, Hackett NR, Crystal RG, Mastrangeli A. Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype. Hum Gene Ther. 1997;8:99–109.[Medline] [Order article via Infotrieve]
    
18. Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C, Selby PJ. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer. 1998;77:956–964.[Medline] [Order article via Infotrieve]
    
19. Campeau L. Grading of angina pectoris. Circulation. 1976;54:522–523. Letter.[Medline] [Order article via Infotrieve]
    
20. Kligfield P, Hochreiter C, Kramer H, Devereux RB, Niles N, Kramer-Fox R, Borer JS. Complex arrhythmias in mitral regurgitation with and without mitral valve prolapse: contrast to arrhythmias in mitral valve prolapse without mitral regurgitation. Am J Cardiol. 1985;55:1545–1549.[Medline] [Order article via Infotrieve]
    
21. Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human subjects. J Am Coll Cardiol. 1985;5:587–592.[Abstract]
    
22. Williams KA, Taillon LA. Reversible ischemia in severe stress technetium 99m-labeled sestamibi perfusion defects assessed from gated single-photon emission computed tomographic polar map Fourier analysis. J Nucl Cardiol. 1995;2:199–206.[Medline] [Order article via Infotrieve]
    
23. Okin PM, Kligfield P. Heart rate adjustment of ST segment depression and performance of the exercise electrocardiogram: a critical evaluation. J Am Coll Cardiol. 1995;25:1726–1735.[Abstract]
    
24. Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 1997;277:48–50.[Free Full Text]
    
25. Laitinen M, Hartikainen J, Eranen J, Kiviniemi M, Hiltunen MO, Laakso M, Ya-Herttuala S. Catheter-mediated VEGF gene transfer to human coronary arteries after angioplasty: safety results from phase I Kuopio angioplasty gene transfer trial (KAT trial). Circulation. 1998;98(suppl I):I-322. Abstract.
    
26. Harvey B-G, Hackett N-R, El-Saw YT, Rosengart TK, Hirschowitz EA, Lieberman MO, Lesser ML, Crystal RG. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol. In press.
    
27. Ohwada A, Hirschowitz EA, Crystal RG. Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver. Hum Gene Ther. 1996;7:1567–1576.[Medline] [Order article via Infotrieve]
    
28. Gao GP, Yang Y, Wilson JM. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol. 1996;70:8934–8943.[Abstract]
    
29. Worgall S, Wolff G, Falck-Pedersen E, Crystal RG. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther. 1997;8:37–44.[Medline] [Order article via Infotrieve]
    
30. Wilson JM. Adenoviruses as gene-delivery vehicles. N Engl J Med. 1996;334:1185–1187.[Free Full Text]
    
31. Hariawala MD, Horowitz JJ, Esakof D, Sheriff DD, Walter DH, Keyt B, Isner JM, Symes JF. VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts. J Surg Res. 1996;63:77–82.This report summarizes a phase I clinical experience with a gene-therapy strategy with an E1^-E3^- adenovirus (Ad) gene-transfer vector expressing the human vascular endothelial growth factor (VEGF) 121 cDNA (Ad_GVVEGF121.10) to induce therapeutic angiogenesis in the myocardium of individuals with clinically significant coronary artery disease. The data are consistent with the concept that direct myocardial administration of Ad_GVVEGF121.10 to individuals with clinically significant coronary artery disease appears to be well tolerated, and initiation of phase II evaluation of this therapy is warranted.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				T. Kinnaird, E. Stabile, S. Zbinden, M.-S. Burnett, and S. E. Epstein Cardiovascular risk factors impair native collateral development and may impair efficacy of therapeutic interventions Cardiovasc Res, May 1, 2008; 78(2): 257 - 264. [Abstract] [Full Text] [PDF]

				D. Ricci, A. A. Mennander, L. D. Pham, V. P. Rao, N. Miyagi, G. W. Byrne, S. J. Russell, and C. G.A. McGregor Non-invasive radioiodine imaging for accurate quantitation of NIS reporter gene expression in transplanted hearts Eur. J. Cardiothorac. Surg., January 1, 2008; 33(1): 32 - 39. [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]

				T. D. Henry, C. L. Grines, M. W. Watkins, N. Dib, G. Barbeau, R. Moreadith, T. Andrasfay, and R. L. Engler Effects of Ad5FGF-4 in Patients With Angina: An Analysis of Pooled Data From the AGENT-3 and AGENT-4 Trials J. Am. Coll. Cardiol., September 11, 2007; 50(11): 1038 - 1046. [Abstract] [Full Text] [PDF]

				J. C. Wu, F. M. Bengel, and S. S. Gambhir Cardiovascular Molecular Imaging Radiology, August 1, 2007; 244(2): 337 - 355. [Abstract] [Full Text] [PDF]

				H. Lu, X. Xu, M. Zhang, R. Cao, E. Brakenhielm, C. Li, H. Lin, G. Yao, H. Sun, L. Qi, et al. Combinatorial protein therapy of angiogenic and arteriogenic factors remarkably improves collaterogenesis and cardiac function in pigs PNAS, July 17, 2007; 104(29): 12140 - 12145. [Abstract] [Full Text] [PDF]

				S. M. Vartanian and R. Sarkar Therapeutic Angiogenesis Vascular and Endovascular Surgery, July 1, 2007; 41(3): 173 - 185. [Abstract] [PDF]

				M. L. Springer, A. Banfi, J. Ye, G. von Degenfeld, P. E. Kraft, S. A. Saini, N. K. Kapasi, and H. M. Blau Localization of vascular response to VEGF is not dependent on heparin binding FASEB J, July 1, 2007; 21(9): 2074 - 2085. [Abstract] [Full Text] [PDF]

				S. Rajagopalan, J. Olin, S. Deitcher, A. Pieczek, J. Laird, P. M. Grossman, C. K. Goldman, K. McEllin, R. Kelly, and N. Chronos Use of a Constitutively Active Hypoxia-Inducible Factor-1{alpha} Transgene as a Therapeutic Strategy in No-Option Critical Limb Ischemia Patients: Phase I Dose-Escalation Experience Circulation, March 13, 2007; 115(10): 1234 - 1243. [Abstract] [Full Text] [PDF]

				C. Kim, R.-K. Li, G. Li, Y. Zhang, R. D. Weisel, and T. M. Yau Effects of Cell-Based Angiogenic Gene Therapy at 6 Months: Persistent Angiogenesis and Absence of Oncogenicity Ann. Thorac. Surg., February 1, 2007; 83(2): 640 - 646. [Abstract] [Full Text] [PDF]

				N. Inoue, T. Kondo, K. Kobayashi, M. Aoki, Y. Numaguchi, M. Shibuya, and T. Murohara Therapeutic Angiogenesis Using Novel Vascular Endothelial Growth Factor-E/Human Placental Growth Factor Chimera Genes Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 99 - 105. [Abstract] [Full Text] [PDF]

				T. Ishikawa, M. Eguchi, M. Wada, Y. Iwami, K. Tono, H. Iwaguro, H. Masuda, T. Tamaki, and T. Asahara Establishment of a Functionally Active Collagen-Binding Vascular Endothelial Growth Factor Fusion Protein In Situ Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 1998 - 2004. [Abstract] [Full Text] [PDF]

				R. S. Ripa, Y. Wang, E. Jorgensen, H. E. Johnsen, B. Hesse, and J. Kastrup Intramyocardial injection of vascular endothelial growth factor-A165 plasmid followed by granulocyte-colony stimulating factor to induce angiogenesis in patients with severe chronic ischaemic heart disease Eur. Heart J., August 1, 2006; 27(15): 1785 - 1792. [Abstract] [Full Text] [PDF]

				F. W. Sellke, R. Laham, E. J. Suuronen, and M. Ruel Angiogenesis for the treatment of inoperable coronary disease: the future. Seminars in Cardiothoracic and Vascular Anesthesia, June 1, 2006; 10(2): 184 - 188. [Abstract] [PDF]

				M. A. Nordlie, L. E. Wold, B. Z. Simkhovich, C. Sesti, and R. A. Kloner Molecular Aspects of Ischemic Heart Disease: Ischemia/Reperfusion-Induced Genetic Changes and Potential Applications of Gene and RNA Interference Therapy Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2006; 11(1): 17 - 30. [Abstract] [PDF]

				M. Hanif, A. Patel, and J. Dunning Might gene therapy offer symptomatic relief for patients with 'no option' angina? Interactive CardioVascular and Thoracic Surgery, December 1, 2005; 4(6): 627 - 632. [Abstract] [Full Text] [PDF]

				C. R. Bridges, K. Gopal, D. E. Holt, C. Yarnall, S. Cole, R. B. Anderson, X. Yin, A. Nelson, B. W. Kozyak, Z. Wang, et al. Efficient myocyte gene delivery with complete cardiac surgical isolation in situ J. Thorac. Cardiovasc. Surg., November 1, 2005; 130(5): 1364 - 1364. [Abstract] [Full Text] [PDF]

				Y. Tsutsumi and D. W. Losordo Double Face of VEGF Circulation, August 30, 2005; 112(9): 1248 - 1250. [Full Text] [PDF]

				D. Hou, E. A.-S. Youssef, T. J. Brinton, P. Zhang, P. Rogers, E. T. Price, A. C. Yeung, B. H. Johnstone, P. G. Yock, and K. L. March Radiolabeled Cell Distribution After Intramyocardial, Intracoronary, and Interstitial Retrograde Coronary Venous Delivery: Implications for Current Clinical Trials Circulation, August 30, 2005; 112(9_suppl): I-150 - I-156. [Abstract] [Full Text] [PDF]

				M. Gyongyosi, A. Khorsand, S. Zamini, W. Sperker, C. Strehblow, J. Kastrup, E. Jorgensen, B. Hesse, K. Tagil, H. E. Botker, et al. NOGA-Guided Analysis of Regional Myocardial Perfusion Abnormalities Treated With Intramyocardial Injections of Plasmid Encoding Vascular Endothelial Growth Factor A-165 in Patients With Chronic Myocardial Ischemia: Subanalysis of the EUROINJECT-ONE Multicenter Double-Blind Randomized Study Circulation, August 30, 2005; 112(9_suppl): I-157 - I-165. [Abstract] [Full Text] [PDF]