Gene transfer for therapeutic angiogenesis in cardiovascular diseases

Peter R. Vale1, Douglas W. Losordo2 Departments of 1Vascular Medicine and 2Cardiology, St Elizabeth’s Medical Center, Tuft’s University School of Medicine, Boston, Mass, USA
Correspondence: Dr Douglas W. Losordo, St Elizabeth’s Medical Center, 736 Cambridge St, Boston, MA 02135, USA. Tel: +1 617 7893346, fax: +1 617 7895069, e-mail: douglas.losordo@tufts.edu

Introduction
Cardiovascular atherosclerotic diseases remain leading causes of morbidity and mortality worldwide. Despite the significant progress that has been made in the management of these diseases using medical, surgical, and percutaneous therapies over the last three decades, there remains a significant population of patients who are not optimal candidates for surgical or percutaneous revascularization.
Substantial research has focused on the administration of angiogenic growth factors, either as recombinant protein or by gene transfer, to promote the development of
supplemental collateral blood vessels that will constitute endogenous bypass conduits around occluded native arteries — a strategy termed “therapeutic angiogenesis.” While many cytokines have angiogenic activity, the best studied both in animal models and
clinical trials are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).
This review will discuss gene transfer strategies for therapeutic angiogenesis in critical limb and myocardial ischemia.

Neovascularization
The strategies of gene transfer for therapeutic angiogenesis are ultimately based upon the findings of Folkman [1], who suggested that the establishment and maintenance of a vascular supply is an absolute requirement for growth of normal or neoplastic tissue as a result of two main processes, vasculogenesis and angiogenesis, and that such neovascularization involves angiogenic growth factors. Vasculogenesis is the de novo, in situ differentiation of endothelial cells from mesodermal precursors in the embryo by association of endothelial progenitor cells or angioblasts and their subsequent reorganization into a primary capillary plexus [2], and was previously considered to be restricted to embryonic development. Angiogenesis is the formation of new blood vessels from pre-existing blood vessels, induced by the proliferation and migration of pre-existing, fully differentiated endothelial cells resident within parent vessels in response to stimuli such as hypoxia, ischemia, mechanical stretch, and inflammation [3, 4], and was thought to be exclusively responsible for postnatal neovascularization
More recent evidence suggests that the basis for native, as well as therapeutic, neovascularization is not restricted to angiogenesis but encompasses both embryonic processes. The demonstration that bone marrow-derived endothelial progenitor cells increase in number in response to tissue ischemia [5], are home to and incorporate into the foci of neovascularization in adult animals [6], and can augment collateral development following ex vivo expansion and transplantation [7] suggests that neovascularization in the adult is not restricted to angiogenesis but involves “postnatal vasculogenesis.”
Angiogenic growth factor-induced neovascularization encompasses a range of vessel calibers, from medium-sized arteries visualized by angiography to increased capillary density demonstrated by postmortem histology. A proportion of newly recognized medium-sized arteries may develop as a result of “arteriogenesis” or in situ proliferation of pre-existing arteriolar connections into larger collateral vessels by remodeling [8]; it is unknown whether such remodeling occurs as a direct result of growth factor modulation or as a flow-mediated maturation of these collateral conduits.

Therapeutic angiogenesis
Angiogenic cytokines may be administered as recombinant protein or as genes encoding these proteins. Protein therapy remains the more conventional approach, but its discussion is beyond the scope of this review. Nevertheless, recombinant protein is usually administered systemically and is therefore limited by potential adverse effects of the high plasma concentrations required to achieve adequate tissue uptake.
Gene transfer is the introduction of genetic material into somatic cells of an organism with the aim of achieving high levels of sustained gene expression without provoking adverse host reactions. There are two major categories of gene transfer systems, viral and nonviral. The most commonly used viral vectors for gene transfer are adenovirus and retrovirus. The nonviral methods include introduction of naked DNA into the target area and the use of liposomes.
Ischemic muscle represents a promising target for gene transfer. Striated and cardiac muscles have been shown to take up and express naked plasmid DNA as well as transgenes incorporated into viral vectors. Moreover, previous studies have shown that the transfection efficiency of intramuscular gene transfer is augmented more than fivefold when the injected muscle is ischemic [9, 10]. However, while viral vectors may enhance transfection efficiency and thus yield higher levels of gene expression, in vitro [11] and in vivo [12] models demonstrated that low-efficiency, but site-specific, transfection (successful transfection in <1% of cells) with a gene (plasmid DNA) encoding a secreted protein (eg, VEGF) may overcome the handicap of inefficient transfection by secreting adequate protein to achieve local levels with physiologically meaningful biological effects, therefore achieving therapeutic effects not realized by transfection with genes encoding proteins that remain intracellular (eg, basic fibroblast growth factor [bFGF]). Furthermore, unlike viral vectors, plasmid DNA does not induce inflammation.

Critical limb ischemia
In a large proportion of patients with critical limb ischemia, the distribution and extent of the arterial occlusive disease make percutaneous or surgical revascularization impossible. The consensus statement of the European Working Group on Critical Limb Ischaemia [13] states that no medical treatment has been shown to alter the natural history of critical limb ischemia. In addition, quality-of-life indices for these patients are similar to those in patients with terminal stages of malignancy. Despite the morbidity and mortality associated with amputation, it is often chosen as first-line therapy. Consequently, the need for alternative treatment strategies in patients with critical limb ischemia is compelling.

VEGF gene transfer in peripheral ischemia
Evidence that VEGF stimulates angiogenesis in vivo has been obtained in experiments performed on rat and rabbit cornea, the chorioallantoic membrane, and the rabbit bone graft model [14, 15]. Initial preclinical studies established that the angiogenic activity of VEGF is sufficiently potent to achieve therapeutic benefit; augmentation of angiographically visible collateral vessels and histologically identifiable capillaries were demonstrated in rabbits with severe, unilateral, hindlimb ischemia [16, 17]. Subsequently, angiographic and histologic evidence of angiogenesis was demonstrated following intra-arterial gene transfer of naked plasmid DNA encoding VEGF (phVEGF165) in humans [18].
However, intra-arterial delivery has several inherent limitations that undermine successful gene transfer for critical limb ischemia. In the case of naked DNA, ie, DNA unassociated with viral or other adjunctive vectors, cellular uptake is virtually nil when the transgene is directly injected into the arterial lumen, presumably due to prompt degradation by circulating nucleases. In addition, the diffuse distribution of neointimal thickening and/or extensive calcific deposits may limit gene transfer to the smooth muscle cells of the arterial media [19].
Preclinical studies subsequently established the feasibility of site-specific intramuscular gene transfer of VEGF in critical limb ischemia to promote therapeutic angiogenesis. Meaningful biological outcomes were observed following phVEGF165 gene transfer by direct injection into skeletal muscle of ischemic rabbit hindlimbs [10, 20], as evidenced by increased hindlimb blood pressure ratio, increased Doppler-derived iliac flow, enhanced neovascularity by angiography, and increased capillary density at necropsy.
That intramuscular VEGF gene transfer might be utilized to successfully accomplish therapeutic angiogenesis in patients with critical limb ischemia was demonstrated in patients with nonhealing ischemic ulcers and/or rest pain by intramuscular injection of 4000 mg phVEGF165 [21]. Gene expression was documented by a transient increase in serum levels of VEGF monitored by ELISA. Therapeutic benefit was demonstrated by regression of rest pain and/or improved limb integrity, increased pain-free walking time and ankle-brachial index, newly visible collateral vessels by digital subtraction angiography, and qualitative evidence of improved distal flow by magnetic resonance imaging.
Subsequent clinical trials with phVEGF165 have utilized randomized (blinded) intramuscular injections in 55 patients (ages 24 to 84 years, mean 56.7 years) with ischemic rest pain (n = 14) or ischemic ulcers (n = 41). Evidence of clinical improvement was observed in 13/14 (72%) patients with rest pain alone and 26/41 (63%) patients with ischemic ulcers over a follow-up period of 4 to 36 months. For the total cohort of 55 patients, a favorable clinical outcome was achieved in 65.5%. Multiple logistic regression analysis identified rest pain and age <50 years as significant (P £0.05) predictors of a favorable clinical outcome. Diabetes, smoking, hyperlipidemia, hypertension, and phVEGF165 dose were not predictors of clinical outcome [22]. Complications in these patients have been limited to lower extremity edema that developed in approximately one-third of patients [23].
A similar treatment strategy was used in 11 patients with Buerger’s disease presenting with critical limb ischemia, nine of whom were successfully treated [24]. These patients had resolution of nocturnal rest pain and healing of foot and/or leg ulcers. The ankle-brachial index increased by greater than 0.1 and newly formed collateral vessels were seen on magnetic resonance and serial contrast angiography.
Preclinical studies from our laboratory demonstrated that VEGF-2 could promote angiogenesis in a rabbit hindlimb ischemia model and stimulate the release of nitric oxide from endothelial cells [25]. Subsequently, randomized, double-blind, placebo-controlled, dose-escalating trials have commenced to investigate the therapeutic potential of VEGF-2 gene transfer in patients with critical limb ischemia. A total of 46 patients with critical limb ischemia have thus far been randomized on a 3:1 (treatment:placebo) basis to receive saline or plasmid VEGF-2 as naked DNA injected directly into ischemic lower extremity muscles; of 46 patients, 21 had rest pain alone, and 25 had ischemic ulcers ± rest pain. The results of this phase I trial are currently pending.

Myocardial ischemia
For patients in whom antianginal medications fail to provide sufficient symptomatic relief, other interventions such as angioplasty or bypass surgery may be required. While both types of intervention have been shown to be effective for various types of patients, a considerable group of patients may not be candidates for either intervention due to the diffuse nature of their coronary artery disease. Moreover, there are many patients in whom recurrent narrowing and/or occlusion of bypass conduits after initially successful surgery has left the patient again symptomatic with no further option for conventional revascularization.
For the purposes of myocardial angiogenesis, angiogenic cytokines have been administered via a wide variety of routes that include intravenous, intracoronary, transepicardial at time of bypass surgery or via thoracotomy, intrapericardial or periadventitial at time of bypass surgery, and, most recently, transendocardial by catheter.

Preclinical studies with VEGF
Following proof-of-principle demonstration that cytokine gene transfer could be used to promote angiogenesis in humans with critical limb ischemia, we extrapolated this strategy to myocardial ischemia. Direct myocardial gene transfer of phVEGF165 [26, 27] or VEGF-2 [28] via a minimally invasive chest wall incision in a swine model of chronic myocardial ischemia resulted in enhanced collateral vessel-filling and improved perfusion to ischemic myocardium by colored microspheres. Intramyocardial injection of adenovirus encoding VEGF121 via thoracotomy in a pig ameroid model improved collateral perfusion and function [29, 30]. Intracoronary adenoviral gene delivery produced much lower gene and VEGF levels in the myocardium with poor localization [30]. Pericardial delivery of adenovirus encoding VEGF165 in a dog model did not increase collateral flow [31].
Preliminary preclinical studies in swine, utilizing a previously described navigation system and catheter mapping technology (NOGA™) integrated with an injection catheter (Biosense Webster, Warren, NJ, USA) to deliver plasmid DNA encoding a reporter gene to the myocardium [32], established that percutaneous myocardial gene transfer could be successfully achieved in normal and ischemic myocardium in a relatively site-specific fashion without significant morbidity or mortality. Similar findings were demonstrated by a study utilizing adenoviral-assisted gene transfer of a reporter gene [33]. Safe and effective gene transfer was also demonstrated in studies utilizing catheter-based delivery of naked plasmid DNA encoding VEGF-1 and VEGF-2 [26], as evidenced by the presence of plasmid DNA in myocardial tissue by PCR, absence of VEGF protein or plasmid in remote organs and absence of hemodynamic changes, sustained ventricular arrhythmias, or ECG evidence of infarction. Objective evidence of reduced ischemia (reduced ischemic area on NOGA™ mapping) was documented in all VEGF-transfected animals, whereas no improvement was seen in control animals. Therefore, the mapping capabilities of the NOGA™ system utilized in these studies were useful for demonstrating that gene expression could be directed to predetermined LV sites, indicating that this technique clearly may be advantageous for avoiding gene transfer to sites of myocardial scar as well as for accurately relocating the tip of an injection catheter to areas of myocardial ischemia (or hibernating myocardium) where gene expression may be potentially optimized.

Preclinical studies with FGF
Intracoronary FGF-2 protein improved myocardial perfusion in myocardial ischemia in both the dog [34, 35] and the pig [36], but recombinant FGF-1 protein was ineffective in the dog [37]. Experience with FGF gene transfer is more limited. Single-dose intramuscular injection of naked DNA encoding FGF-1 and intracoronary adenoviral transfer of the FGF-5 gene have each been shown to improve flow in the rabbit hindlimb [38] and porcine myocardium [39], respectively.

Clinical trials of direct myocardial VEGF gene transfer
Published studies of VEGF gene transfer for therapeutic angiogenesis in human subjects have thus far been limited to phase I, dose-escalating, nonrandomized trials involving naked plasmid DNA and adenoviral vectors. Patients in these trials generally have severe angina refractory to medical therapy, demonstrate ongoing myocardial ischemia, and are unsuitable for conventional revascularization.
As a result of the above animal experiments of VEGF plasmid DNA gene transfer, our center initiated a phase I, dose-escalating, open-label, clinical study of myocardial gene transfer of phVEGF165 as sole therapy (ie, without PTCA or CABG surgery) in 30 “no-option” patients [40, 41], administered by direct injection via a limited anterior thoracotomy under guidance of continuous transesophageal echocardiographic monitoring [42]. No perioperative complications were experienced. Gene expression was documented by a transient but significant increase in plasma levels of VEGF monitored by ELISA assay. Clinical improvement was demonstrated by a reduction in sublingual nitrate use and anginal episodes per week, and an increase in exercise time by 98 seconds. Evidence of reduced ischemia on SPECT-sestamibi myocardial perfusion scanning was documented by a significant reduction in both mean stress and rest perfusion/ischemia scores. Importantly, there was improvement after gene transfer in resting defects, suggesting that these pre-existing resting defects constitute foci of hibernating viable myocardium [43–45], which have improved contractile activity as a result of therapeutic neovascularization. This observation was supported by the findings of electromechanical mapping utilized in the final 13 consecutive patients. Resting perfusion defects on the SPECT images corresponded to areas with ischemic characteristics (reduced wall motion with preserved viability) on the endocardial maps. Foci of ischemia identified preoperatively showed significant improvement in these endocardial wall motion abnormalities 60 days after gene transfer [46].
Similar favorable experience has been realized in an open-label, dose-escalating, multicenter, clinical trial of VEGF-2 plasmid DNA gene transfer in 30 patients with end-stage coronary artery disease and refractory class 3 or 4 angina. In all patients, there were no procedural adverse events, although there was one death 20 hours after surgery. Twelve months after gene transfer, the mean number of anginal episodes and nitrate tablets consumed per week decreased significantly, 25/29 patients (86%) had improved by 2 or more angina classes, and mean duration of exercise increased by more than 2 minutes (unpublished data).
The only other reported study of direct myocardial VEGF gene transfer was with adenoviral-assisted VEGF121 injection to patients undergoing bypass graft surgery (n = 15), or as sole therapy via a minithoracotomy (n = 6). Symptoms and exercise duration improved in both bypass surgery and sole therapy groups, but stress-induced nuclear perfusion images remained unchanged. The data in this study are consistent with the concept that adenovirus VEGF121 appears to be well tolerated in patients with advanced coronary disease.
This early experience with myocardial VEGF gene transfer, while encouraging from the standpoint of therapeutic angiogenesis and gene therapy, leaves many issues unresolved. Optimizing the anatomic site, number, and dose of direct myocardial injections will require further investigation. The strategy of gene therapy alone administered via a minithoracotomy does not permit randomization against placebo (untreated controls) or clinical testing of alternative dosing regimens including multiple treatments.

Clinical applications of catheter-based myocardial VEGF gene transfer
While successful intravascular [18], pericardial [47], and intramuscular [21] gene transfer have all been performed using minimally invasive delivery techniques, all of the clinical trials involving myocardial gene transfer have to date required an operative procedure.
As a result of preclinical studies, we initiated a pilot study of percutaneous, catheter-based VEGF-2 DNA gene transfer or a sham procedure guided by the NOGA™ mapping system in six patients with nonrevascularizable symptomatic myocardium [48]. VEGF-2-transfected patients reported significant reductions in weekly anginal episodes and nitrate tablet consumption 12 months after gene transfer. In contrast, while blinded patients randomized to the control group reported an initial reduction in these parameters, this changed clinical profile was not sustained beyond 30 days, suggesting that the continued reduction in angina in the VEGF-2-treated group was not a placebo effect. The symptomatic improvement was again accompanied by objective evidence of improved myocardial perfusion by both SPECT-sestamibi perfusion scanning and electromechanical mapping [48].
While the clinical findings of this pilot trial concerning efficacy are similarly encouraging, the number of patients and the single-blind design preclude firm conclusions in this regard. Consequently, a multicenter, randomized, double-blind, placebo-controlled trial of catheter-based VEGF-2 gene transfer is underway that has thus far enrolled 19 patients. There have been no complications associated with a total of 150 injections among the 25 patients given either VEGF-2 or placebo in these two studies.
This preliminary experience suggests that it is feasible to replace currently employed operative approaches with minimally invasive techniques for applications of cardiovascular gene therapy designed to target myocardial function and perfusion. Such an approach may have at least three advantages compared with an operative approach. First, it potentially allows more selective delivery of the transgene to targeted ischemic zones, including sites that are less accessible by a minithoracotomy. Second, the catheter-based approach, because it obviates the need for general anesthesia and operative dissection through adhesions related to placement of previous bypass conduits, facilitates placebo-controlled, double-blind testing of myocardial gene transfer. Third, the intervention can be performed as an outpatient procedure and repeated if necessary.

Myocardial gene transfer with FGF
Clinical experience with FGF gene transfer for myocardial angiogenesis is limited. Perivascular FGF-2 (bFGF) or placebo contained within heparin alginate microcapsules was implanted in the subepicardial fat in nongraftable myocardial territory in patients undergoing CABG with viable and ischemic myocardium [49]. In this study, there was relatively high perioperative mortality and morbidity, attributed to the advanced nature of coronary artery disease. However, preliminary data suggested improvement in angina, myocardial perfusion, and regional function by MRI in the bFGF group.

Arterial gene therapy for inhibiting restenosis in patients with claudication undergoing superficial femoral artery (SFA) angioplasty
Superficial femoral artery (SFA) stenosis represents one of the most common sites of peripheral vascular obstruction. Percutaneous transluminal angioplasty (PTA) has been used widely and successfully to treat atherosclerotic obstructions in the peripheral and coronary circulations. However, restenosis following angioplasty of the SFA/popliteal artery continues to be a vexing and, consequently, expensive complication of this otherwise efficacious intervention. While acute procedural success for percutaneous revascularization of lesions in the SFA using conventional guidewires and standard PTA is well in excess of 90%, published reports have established that restenosis may complicate the clinical course of as many as 60% of patients undergoing PTA for SFA stenosis and/or occlusion. Previous strategies to limit the development of restenosis by nonmechanical means have not proved effective. Treatment strategies aimed at specifically restoring endothelial integrity have not been previously explored for restenosis prevention. Animal studies demonstrated that administration of mitogens, such as VEGF, that promote endothelial cell migration and/or proliferation might achieve acceleration of re-endothelialization and thereby reduce intimal thickening [50–53].
A phase I clinical trial was initiated utilizing naked plasmid VEGF165 DNA applied to the hydrogel coating of an angioplasty balloon catheter and transfected at the site of PTA. The objectives were to document the safety and efficacy of this strategy to accelerate re-endothelialization at the site of PTA-induced endothelial disruption as a novel means to inhibit restenosis in patients with claudication due to SFA obstruction.
Thus far, 20 patients have received arterial VEGF (13 males, mean age 69 years). Gene expression was documented by a rise in serum levels of VEGF. At latest follow-up (up to 18 months), mean claudication time increased from 2 to 5 minutes, mean Rutherford class (all were class 3 at baseline) improved by 2 or more (10 patients were asymptomatic and five patients were class 1), and mean ankle-brachial index increased from 0.70 to 0.89. Five patients had evidence of restenosis at angiography performed 6 to 12 months after gene transfer. Target vessel revascularization was required in all five patients. Histology from three out of four patients to undergo directional atherectomy at the time of repeat revascularization for restenosis demonstrated active smooth muscle cell proliferation and high levels of proliferating cell nuclear antigen indicating extensive proliferative activity. In the remaining 15 patients, mean SFA stenosis dropped from 82% to 32% at an average of 9 months following gene transfer. These results were supported by intravascular ultrasound findings at the time of follow-up angiography.
This preliminary study has suggested that gene transfer designed to accelerate re-endothelialization at the site of PTA-induced endothelial disruption can be safely performed. Furthermore, 5 out of 20 patients (25%) required target vessel revascularization for angiographic and ultrasound evidence of restenosis, suggesting that this may be an effective strategy to prevent restenosis following SFA angioplasty. Importantly, no evidence of accelerated atherosclerosis or an increase in the restenosis rate was observed following gene transfer.

Potential safety concerns
Many angiogenic factors are known to be involved in tumor growth secondary to enhancing angiogenesis. Hence, in theory, angiogenic growth factors may lead to the development of tumors which may be too small to be recognized. Even so, there are neither in vitro nor in vivo data to suggest that VEGF increases the risk of neoplastic growth and/or metastases, although longer term follow-up will be required to address this issue in clinical trials. Nevertheless, one must be vigilant about the possibility of cancer in patients treated with angiogenic growth factors. In addition, concerns regarding the development of angiomata were raised in studies involving mice [54] or rats [55] treated with transduced myoblasts or supraphysiologic doses of plasmid DNA, respectively. Importantly, no other preclinical or clinical reports, including those using adenoviral vectors, have described this complication.
It is theoretically possible that VEGF may exacerbate proliferative and/or hemorrhagic retinopathy in patients with diabetes in view of the high VEGF levels demonstrated in the ocular fluid of patients with active proliferative retinopathy leading to loss of vision [56]. To date, this adverse effect of therapeutic angiogenesis has not been observed. The local delivery of naked plasmid DNA encoding VEGF-1 or VEGF-2 to more than 100 patients (one-third with diabetes and/or remote retinopathy) treated at our institution with up to 4-year follow-up did not affect the visual acuity or funduscopic findings, as evidenced by serial funduscopic examinations before and after gene transfer by an independent group of retinal specialists.
Experiments in transgenic mice engineered to overexpress VEGF ± angiopoietin have demonstrated lethal permeability-enhancing effects of VEGF [57]. However, even though VEGF has been reported to cause local edema, which manifests as pedal edema in patients treated with VEGF for critical limb ischemia, it responds well to treatment with diuretics [23].
Therapies with recombinant proteins have been noted to produce hypotension [58, 59], particularly when used systemically and at higher doses, due to the fact that VEGF upregulates nitric oxide synthesis [60, 61]; this complication, however, has never been described following gene transfer in either animals or humans.
Another concern stems from the recent demonstration that inhibitors of angiogenesis tested in an apolipoprotein E-deficient mouse model of atherosclerosis inhibited plaque growth and intimal neovascularization [62]. However, data available from four separate animal studies [50–53] and two clinical studies of human subjects [63, 64] fail to support the notion that accelerated atherosclerosis is a likely consequence of administering angiogenic cytokines; the outcome, in fact, is quite the opposite, in that administration of VEGF led to a statistically significant reduction in intimal thickening due to accelerated re-endothelialization, thereby refuting the notion that acceleration of atherosclerosis will be a consequence of VEGF-induced stimulation of angiogenesis.

Conclusions
The current clinical strategies employed for critical limb and chronic myocardial ischemia constitute an extrapolation from initial applications of gene transfer to animal models with limb ischemia utilizing the 165-amino acid isoform of the VEGF-1 gene. These results, however, likely have generic implications for strategies of therapeutic neovascularization using alternative candidate genes, vectors, and delivery strategies; all of these are being actively studied in ongoing clinical trials. Furthermore, the relative merits of gene transfer versus recombinant protein administration remain to be clarified.
It is clear that site-specific VEGF gene transfer can be used to achieve physiologically meaningful therapeutic modulation of vascular disorders and, specifically, that intramuscular injection of naked plasmid DNA achieves constitutive overexpression of VEGF sufficient to induce therapeutic angiogenesis in selected patients with critical limb ischemia. Furthermore, at this early stage of clinical trials into myocardial gene therapy, it has been shown that direct myocardial gene transfer utilizing different doses of naked plasmid DNA encoding VEGF165 and VEGF-2 can be performed safely and this approach augments myocardial perfusion. Ongoing clinical studies will determine the potential for neovascularization gene therapy to be performed by nonsurgical, catheter-based delivery, although early results are encouraging from a therapeutic standpoint.
For the most part, clinical studies of therapeutic angiogenesis have been restricted to patients with myocardial or limb ischemia who have no other options. Although this is the group to target in the near future, it is not difficult to foresee a time when the sizeable population of patients who undergo bypass surgery but who are not optimal candidates for that procedure may be eligible for therapeutic angiogenesis, which might be
performed at an earlier stage of disease and thus provide a greater possibility of a successful outcome.

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Baumgartner I, Rauh G, Pieczek A, Wuensch D, Magner M, Kearney M, Schainfeld R, Isner JM.

St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

BACKGROUND: Vascular endothelial growth factor (VEGF) promotes angiogenesis and vascular permeability. The extent to which VEGF may cause tissue edema in humans has not been established. OBJECTIVE: To evaluate patients undergoing VEGF gene transfer for evidence of lower-extremity edema. DESIGN: Prospective consecutive case series. SETTING: Hospital outpatient clinic. PATIENTS: 62 patients with critical limb ischemia and 28 patients with claudication. INTERVENTION: Gene transfer of VEGF DNA. MEASUREMENTS: Semiquantitative analysis of lower-extremity edema. RESULTS: Lower-extremity edema was observed in 31 of 90 (34%) patients. Edema was less common in patients with claudication than in those with pain at rest (P = 0.016) or ischemic ulcers (P < 0.001), and it was less common in patients with pain at rest than in those with ischemic ulcers (P= 0.017). Treatment was typically limited to a brief course of oral diuretics. CONCLUSIONS: Vascular endothelial growth factor may enhance vascular permeability in humans. At the doses of plasmid DNA used in this study, lower-extremity edema responded to oral diuretic therapy and did not seem to be associated with serious sequelae.

PMID: 10836914 [PubMed - indexed for MEDLINE]

 
Treatment of thromboangiitis obliterans (Buerger's disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results.

Isner JM, Baumgartner I, Rauh G, Schainfeld R, Blair R, Manor O, Razvi S, Symes JF.

Division of Cardiovascular Research and the Departments of Medicine, Radiology, and Surgery, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass 02135, USA.

PURPOSE: Thromboangiitis obliterans (TAO), or Buerger's disease, a distinct form of vascular occlusive disease that afflicts the peripheral arteries of young smokers, is often characterized by an inexorable downhill course even in patients who discontinue smoking once a stage of critical limb ischemia associated with ulceration or gangrene is reached. As part of a phase I clinical trial to document the safety and efficacy of intramuscular gene transfer of naked plasmid DNA-encoding vascular endothelial growth factor (phVEGF165) in the treatment of critical limb ischemia, we treated TAO in 6 patients. METHODS: Seven limbs in 6 patients (3 men, 3 women; mean age, 33 years; range, 33 to 51 years) who satisfied the criteria for TAO and had signs or symptoms of critical limb ischemia were treated twice, 4 weeks apart, with 2 or 4 mg of phVEGF165, which was administered by direct intramuscular injection at 4 arbitrarily selected sites in the ischemic limb. The gene expression was documented by enzyme-linked immunosorbent assay that was performed on peripheral blood samples. RESULTS: The ulcers that were nonhealing for more than 1 month healed completely in 3 of 5 limbs after the intramuscular phVEGF165 gene therapy. Nocturnal rest pain was relieved in the remaining 2 patients, although both continue to have claudication. The evidence of the improved perfusion to the distal ischemic limb included an increase of more than 0.1 in the ankle brachial index in 3 limbs, an improved flow shown with magnetic resonance imaging in 7 of the 7 limbs, and newly visible collateral vessels shown with serial contrast angiography in 7 of the 7 limbs. The adverse consequences of the phVEGF165 gene transfer were limited to transient ankle or calf edema in 3 of the 7 limbs. Two patients with advanced distal forefoot gangrene ultimately required below-knee amputation despite the evidence of improved perfusion. A histologic section disclosed the classic pathologic findings of TAO. CONCLUSION: Therapeutic angiogenesis with phVEGF165 gene transfer, if instituted before the development of forefoot gangrene, may provide a novel therapy for patients with advanced Buerger's disease that is unresponsive to standard medical or surgical treatment methods.

PMID: 9845647 [PubMed - indexed for MEDLINE]

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

Witzenbichler B, Asahara T, Murohara T, Silver M, Spyridopoulos I, Magner M, Principe N, Kearney M, Hu JS, Isner JM.

Department of Medicine, St. Elizabeth's Medical Center of Boston, Tufts University School of Medicine, Massachusetts 02135, USA.

Recently, vascular endothelial growth factor-C (VEGF-C or VEGF-2) was described as a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3. In vivo data, limited to constitutive overexpression in transgenic mice, have been interpreted as evidence that the growth-promoting effects of VEGF-C are restricted to development of the lymphatic vasculature. The current studies were designed to test the hypothesis that constitutive expression of VEGF-C in adult animals promotes angiogenesis. In vitro, VEGF-C exhibited a dose-dependent mitogenic and chemotactic effect on endothelial cells, particularly for microvascular endothelial cells (72% and 95% potency, respectively, compared with VEGF-A/VEGF-1). VEGF-C stimulated release of nitric oxide from endothelial cells and increased vascular permeability in the Miles assay; the latter effect was attenuated by pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Both VEGFR-2 and VEGFR-3 receptors were shown to be expressed in human saphenous vein and internal mammary artery. The potential for VEGF-C to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model. Ten days after ligation of the external iliac artery, VEGF-C was administered as naked plasmid DNA (pcVEGF-C; 500 microg) from the polymer coating of an angioplasty balloon (n = 8 each) or as recombinant human protein (rhVEGF-C; 500 microg) by direct intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C. Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased to 0.83 +/- 0.03 after pcVEGF-C versus 0.59 +/- 0.04 (P < 0.005) in pGSVLacZ controls and to 0.76 +/- 0.04 after rhVEGF-C versus 0.58 +/- 0.03 (P < 0.01) in control rabbits receiving rabbit serum albumin. Doppler-derived iliac flow reserve was 2.7 +/- 0.1 versus 2.0 +/- 0.2 (P < 0.05) for pcVEGF-C versus LacZ controls and 2.9 +/- 0.3 versus 2.1 +/- 0.2 (P < 0.05) for rhVEGF-C versus albumin controls. Neovascularity was documented by angiography in vivo (angiographic scores: 0.85 +/- 0.05 versus 0.51 +/- 0.02 (P < 0.001) for plasmid DNA and 0.74 +/- 0.08 versus 0.53 +/- 0.03 (P < 0.05) for protein), and capillary density (per mm2) was measured at necropsy (252 +/- 12 versus 183 +/- 10 (P < 0.005) for plasmid DNA and 229 +/- 20 versus 164 +/- 20 (P < 0.05) for protein). In contrast to the results of gene targeting experiments, constitutive expression of VEGF-C in adult animals promotes angiogenesis in the setting of limb ischemia. VEGF-C and its receptors thus constitute an apparently redundant pathway for postnatal angiogenesis and may represent an alternative to VEGF-A for strategies of therapeutic angiogenesis in patients with limb and/or myocardial ischemia.

PMID: 9708799 [PubMed - indexed for MEDLINE]

 
Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping.

Vale PR, Losordo DW, Tkebuchava T, Chen D, Milliken CE, Isner JM.

Division of Cardiovascular Research, St. Elizabeth's Medical Center, and Tufts University School of Medicine, Boston, Massachusetts, USA.

OBJECTIVES: This study investigated the feasibility and safety of percutaneous, catheter-based myocardial gene transfer. BACKGROUND: Direct myocardial gene transfer has, to date, required direct injection via an open thoracotomy. METHODS: Electroanatomical mapping was performed to establish the site of left ventricular (LV) gene transfer. A steerable, deformable 7F catheter with a 27G needle, which can be advanced 3 to 5 mm beyond its distal tip, was then directed to previously acquired map sites, the needle was advanced, and injections were made into the LV myocardium. RESULTS: In two pigs in which methylene blue dye was injected, discretely stained LV sites were observed at necropsy in each pig, corresponding to the injection sites indicated prospectively by the endocardial map. In six pigs in which the injection catheter was used to deliver plasmid using cytomegalovirus promoter/enhancer, encoding nuclear-specific LacZ gene (pCMV-nlsLacZ) (50 microg/ml) to a single LV myocardial region, peak beta-galactosidase activity after five days (relative light units [RLU], mean 135,333+/-28,239, range = 31,508 to 192,748) was documented in the target area of myocardial injection in each pig. Percutaneous gene transfer of pCMV-nlsLacZ (50 microg/ml) was also performed in two pigs with an ameroid constrictor applied to the left circumflex coronary artery, in each pig, peak beta-galactosidase activity after five days (214,851 and 23,140 RLU) was documented at the injection site. All pigs survived until sacrifice, and no complications were observed with either the mapping or the injection procedures. CONCLUSIONS: Percutaneous myocardial gene transfer can be successfully achieved in normal and ischemic myocardium without significant morbidity or mortality. These findings establish the potential for minimally invasive cardiovascular gene transfer.

PMID: 10400018 [PubMed - indexed for MEDLINE]

 
Intramyocardial gene therapy with naked DNA encoding vascular endothelial growth factor improves collateral flow to ischemic myocardium.

Tio RA, Tkebuchava T, Scheuermann TH, Lebherz C, Magner M, Kearny M, Esakof DD, Isner JM, Symes JF.

Department of Surgery and Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA.

Both VEGF protein and VEGF DNA in combination with an adenoviral vector have been shown to enhance collateral formation in a porcine model of chronic myocardial ischemia. We sought to determine whether direct intramyocardial injection of naked DNA encoding for VEGF could similarly improve myocardial perfusion. Initially, 23 nonischemic pigs received either 200 microg of plasmid DNA encoding beta-galactosidase (pCMVbeta, n = 11) or 500 microg of phVEGF165 (n = 12) into four separate sites in the myocardium via a small anterolateral thoracotomy incision in the fourth intercostal space. Two additional groups of pigs received an intramyocardial injection of either phVEGF165 (n = 6) or pCMVbeta (n = 7) 3 to 4 weeks after implantation of an ameroid constrictor around the left circumflex coronary artery. The injections caused no change in heart rate or blood pressure, and no ventricular arrhythmias or histologic evidence of inflammation. VEGF protein was detected by Western blot in VEGF-treated animals, with the strongest bands closest to the injection site. Plasma VEGF concentration (ELISA) increased from 3+/-2 to 27+/-13 pg/ml (p = 0.035) by day 4 after treatment. No increase in VEGF protein was noted in pCMVbeta-treated animals whereas these did stain positive for beta-Gal. Resting myocardial blood flow (colored microspheres) was significantly reduced in the ischemic versus nonischemic territory in control animals (1.07+/-0.05 versus 1.32+/-0.05; p < 0.05) but not VEGF-treated pigs (1.32+/-0.24 versus 1.13+/-0.12; p = NS). Maximal vasodilatation with adenosine significantly increased flow to the ischemic region in VEGF-treated pigs (2.16+/-0.57 versus 1.32+/-0.24; p < 0.05) but not controls (1.31+/-0.05 versus 1.17+/-0.06;p = NS). Collateral filling of the occluded circumflex artery improved in five of six VEGF-treated pigs (mean change in Rentrop score, +1.5). We conclude that direct intramyocardial transfection phVEGF165 is safe and capable of producing sufficient VEGF protein to enhance collateral formation and myocardial perfusion. This approach may offer an alternative therapy for patients with intractable myocardial ischemia not amenable to PTCA or CABG.

PMID: 10609656 [PubMed - indexed for MEDLINE]

28. Vale PR, Tkebuchava T, Milliken CE, Chen D, Symes JF, Isner JM. Percutaneous electromechanical mapping demonstrates efficacy of pVGI.1 (VEGF2) in an animal model of chronic myocardial ischemia [abstract]. Circulation. 1999;100:I-22.
 

 
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.

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.

Department of Cardiothoracic Surgery, New York Hospital-Cornell Medical Center, New York 10021, USA.

OBJECTIVES: Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, can be delivered to targeted tissues by means of a replication-deficient adenovirus (Ad) vector. We hypothesized that direct administration of Ad vector expressing the VEGF121 complementary deoxyribonucleic acid (AdGVVEGF121.10) into regions of ischemic myocardium would enhance collateral vessel formation and improve regional perfusion and function. METHODS: Yorkshire swine underwent thoracotomy and placement of an Ameroid constrictor (Research Instruments & MFG, Corvallis, Ore.) on the circumflex coronary artery. Three weeks later, myocardial perfusion and function were assessed by single photon emission computed tomography imaging (SPECT) with 99mTc-labeled sestamibi and by echocardiography during rest and stress. AdGVVEGF121.10 (n = 7) or the control vector, AdNull (n = 8), was administered directly into the myocardium at 10 sites in the circumflex distribution (10(8) pfu/site). Four weeks later, these studies were repeated and ex vivo angiography was performed. RESULTS: SPECT imaging 4 weeks after vector administration demonstrated significant reduction in the ischemic area at stress in AdGVVEFG121.10-treated animals compared with AdNull control animals (p = 0.005). Stress echocardiography at the same time demonstrated improved segmental wall thickening in AdGVVEGF121.10 animals compared with AdNull control animals (p = 0.03), with AdGVVEGF121.10 animals showing nearly normalized function in the circumflex distribution. Collateral vessel development assessed by angiography was also significantly greater in AdGVVEGF121.10 animals than in AdNull control animals (p = 0.04), with almost complete reconstitution of circumflex filling in AdGVVEGF121.10 animals. CONCLUSIONS: An Ad vector expressing the VEGF121 cDNA induces collateral vessel development in ischemic myocardium and results in significant improvement in both myocardial perfusion and function. Such a strategy may be useful in patients with ischemic heart disease in whom complete revascularization is not possible.

PMID: 9451061 [PubMed - indexed for MEDLINE]

 
Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121.

Lee LY, Patel SR, Hackett NR, Mack CA, Polce DR, El-Sawy T, Hachamovitch R, Zanzonico P, Sanborn TA, Parikh M, Isom OW, Crystal RG, Rosengart TK.

Department of Cardiothoracic Surgery, The New York Hospital-Cornell Medical Center, New York, USA.

BACKGROUND: Adenovirus (Ad) vector-mediated gene therapy strategies have emerged as promising modalities for the "biological revascularization" of tissues. We hypothesized that direct intramyocardial, as opposed to intracoronary, administration of an Ad vector coding for the vascular endothelial growth factor 121 cDNA (Ad(GV)VEGF121.10) would provide highly focal Ad genome levels, and increases in VEGF, ideal for inducing localized therapeutic angiogenesis. METHODS: Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial Ad(GV)VEGF121.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model. RESULTS: Intramyocardial delivery of Ad(GV)VEGF121.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial Ad(GV)VEGF121.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial Ad(GV)VEGF121.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration. CONCLUSIONS: These studies demonstrate that direct intramyocardial administration of Ad(GV)VEGF121.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human trials of gene therapy-mediated cardiac angiogenesis.

PMID: 10654479 [PubMed - indexed for MEDLINE]

 
Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis.

Lazarous DF, Shou M, Stiber JA, Hodge E, Thirumurti V, Goncalves L, Unger EF.

Experimental Physiology and Pharmacology Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. dlazarou@welch.jhu.edu

OBJECTIVE: Angiogenic peptides like VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) have entered clinical trials for coronary artery disease. Attempts are being made to devise clinically relevant means of delivery and to effect site-specific delivery of these peptides to the cardiac tissue, in order to limit systemic side-effects. We characterized the response of the pericardium to delivery of a replication-deficient adenovirus carrying the cDNA for AdCMV.VEGF165, and assessed the effect of pericardial VEGF165 on myocardial collateral development in a canine model of progressive coronary occlusion. METHODS: Ameroid constrictors were placed on the proximal left circumflex coronary artery of mongrel dogs. Ten days later, 6 x 10(9) pfu AdCMV.VEGF165 (n = 9). AdRSV.beta-gal (n = 9), or saline (n = 7) were injected through an indwelling pericardial catheter. Transfection efficiency was assessed by X-gal staining. Pericardial and serum VEGF levels were measured serially by ELISA. Maximal myocardial collateral perfusion was quantified with radiolabeled or fluorescent microspheres 28 days after treatment. RESULTS: In AdRSV.beta-gal-treated dogs, there was extensive beta-gal staining in the pericardium and epicardium, with minimal beta-gal staining in the mid-myocardium and endocardium. Pericardial delivery of AdCMV.VEGF165 resulted in sustained (8-14 day) pericardial transgene expression, with VEGF levels peaking 3 days after infection (> 200 ng/ml) and decreasing thereafter. There was no detectable increase in serum VEGF levels. Maximal collateral perfusion, a principal correlate of collateral development and angiogenesis, was equivalent in all groups. CONCLUSION: Adenoviral-mediated gene transfer is capable of inducing sustained VEGF165 expression in the pericardium; however, locally targeted pericardial VEGF delivery failed to improve myocardial collateral perfusion in this model.

PMID: 10690306 [PubMed - indexed for MEDLINE]

 
Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping.

Vale PR, Losordo DW, Tkebuchava T, Chen D, Milliken CE, Isner JM.

Division of Cardiovascular Research, St. Elizabeth's Medical Center, and Tufts University School of Medicine, Boston, Massachusetts, USA.

OBJECTIVES: This study investigated the feasibility and safety of percutaneous, catheter-based myocardial gene transfer. BACKGROUND: Direct myocardial gene transfer has, to date, required direct injection via an open thoracotomy. METHODS: Electroanatomical mapping was performed to establish the site of left ventricular (LV) gene transfer. A steerable, deformable 7F catheter with a 27G needle, which can be advanced 3 to 5 mm beyond its distal tip, was then directed to previously acquired map sites, the needle was advanced, and injections were made into the LV myocardium. RESULTS: In two pigs in which methylene blue dye was injected, discretely stained LV sites were observed at necropsy in each pig, corresponding to the injection sites indicated prospectively by the endocardial map. In six pigs in which the injection catheter was used to deliver plasmid using cytomegalovirus promoter/enhancer, encoding nuclear-specific LacZ gene (pCMV-nlsLacZ) (50 microg/ml) to a single LV myocardial region, peak beta-galactosidase activity after five days (relative light units [RLU], mean 135,333+/-28,239, range = 31,508 to 192,748) was documented in the target area of myocardial injection in each pig. Percutaneous gene transfer of pCMV-nlsLacZ (50 microg/ml) was also performed in two pigs with an ameroid constrictor applied to the left circumflex coronary artery, in each pig, peak beta-galactosidase activity after five days (214,851 and 23,140 RLU) was documented at the injection site. All pigs survived until sacrifice, and no complications were observed with either the mapping or the injection procedures. CONCLUSIONS: Percutaneous myocardial gene transfer can be successfully achieved in normal and ischemic myocardium without significant morbidity or mortality. These findings establish the potential for minimally invasive cardiovascular gene transfer.

PMID: 10400018 [PubMed - indexed for MEDLINE]

 
Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. Results in normal and ischemic porcine models.

Kornowski R, Leon MB, Fuchs S, Vodovotz Y, Flynn MA, Gordon DA, Pierre A, Kovesdi I, Keiser JA, Epstein SE.

Cardiovascular Research Foundation, Washington Hospital Center, Washington, DC 20010, USA. rxk3@mhg.edu

OBJECTIVES: To test the feasibility of myocardial angiogenic gene expression using a novel catheter-based transendocardial injection system. BACKGROUND: Angiogenesis has been induced by direct injection of growth factors into ischemic myocardium during open-heart surgery. Catheter-based transendocardial injection of angiogenic factors may provide equivalent benefit without need of surgery. METHODS: A new guidance system for intramyocardial therapy utilizes magnetic fields and catheter-tip sensors to locate a position in space and reconstruct three-dimensional left ventricular (LV) electromechanical maps without using fluoroscopy. A retractable 27G needle was coupled with the guidance system for LV transendocardial injection. In 12 pigs, the catheter was used to inject 0.1 ml of methylene-blue (MB) dye and 8 pigs had myocardial injections of adenoviral vector (1 x 10(10) particles per site) containing the LacZ transgene. Ten pigs underwent catheter-based transendocardial injection and six pigs were injected using transepicardial approach with the gene encoding adenovirus vascular endothelial growth factor-121 (Ad.VEGF121; 1 x 10(10) viral particles x 6 sites) and sacrificed at 24 h. Injection sites were identified with ultraviolet light by coinjection of fluorescent beads. RESULTS: Overall, 138 of 152 attempted injection MB tracks (91%) were found after sacrifice. Tissue staining was 7.1+/-2.1 mm in depth and 2.3+/-1.8 mm in width. No animal had pericardial effusion or tamponade. In Ad.LacZ injected animals, gross pathology showed positive staining in injected zones, and histology confirmed positive myocyte staining. Adenovirus vascular endothelial growth factor-121 injected sites showed high levels of VEGF121 production that was of similar magnitude whether injected using the transendocardial (880.4+/-412.2 pg VEGF121/mg protein) or transepicardial (838.3+/-270 pg VEGF121/mg protein) delivery approach (p = 0.62). CONCLUSIONS: Using this magnetic guidance catheter-based navigational system, transgenes can effectively be transfected into designated myocardial sites. Thus, if it is determined that direct intramyocardial injection of angiogenic factors enhances collateral function in patients, this less invasive catheter-based system offers a similar gene delivery efficiency and, thus, may have clear advantages compared with the surgically-based transepicardial injection approach.

PMID: 10732905 [PubMed - indexed for MEDLINE]

 
Effects of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart.

Lazarous DF, Scheinowitz M, Shou M, Hodge E, Rajanayagam S, Hunsberger S, Robison WG Jr, Stiber JA, Correa R, Epstein SE, et al.

Section of Experimental Physiology and Pharmacology, Cardiology Branch, National Institutes of Health, Bethesda, Md 20892.

BACKGROUND: Recently we reported that intracoronary administration of basic fibroblast growth factor (bFGF), a potent angiogenic peptide, increases collateral blood flow in dogs subjected to progressive left circumflex coronary artery (LCx) occlusion. The aim of the present study was to examine the effect of systemically administered bFGF on collateral blood flow and to assess its pharmacokinetics and potential side effects. METHODS AND RESULTS: Forty-seven dogs were subjected to progressive ameroid-induced occlusion of the LCx, an intervention known to induce the development of collateral vessels. In phase I of the investigation, dogs were randomized to receive bFGF 1.74 mg/d (n = 10) or saline (n = 9) as a left atrial injection for 4 weeks. Relative collateral blood flow was assessed serially with radiolabeled microspheres in the conscious state during maximal coronary vasodilatation. Initiation of bFGF treatment was temporally associated with a marked acceleration of collateral development; however, collateral flow in control dogs improved toward the end of the study, approaching that of bFGF-treated dogs at the 38-day end point. Phase II of the investigation was a three-armed study of extended duration to determine whether bFGF caused a sustained increase in collateral function. Dogs were randomized to receive bFGF 1.74 mg/d for 9 weeks (n = 7), bFGF 1.74 mg/d for 5 weeks followed by placebo for 4 weeks (n = 11), or placebo for 9 weeks (n = 10). Relative and absolute collateral blood flow were assessed serially with microspheres during maximal coronary vasodilatation. Between the 10th and 17th days after ameroid placement, bFGF-treated dogs exhibited marked improvement in collateral flow such that maximal collateral conductance exceeded that of controls by 24% at the 5-week crossover point. Final collateral conductance was similar in dogs receiving bFGF for 5 and 9 weeks despite withdrawal of treatment in the former group. bFGF administration was associated with a 21% increase in final collateral conductance as well as a 49% increase in collateral zone vascular density. Prolonged bFGF administration was also associated with a decrease in arterial pressure, moderate thrombocytopenia, and moderate, reversible anemia. CONCLUSIONS: Systemic administration of bFGF enhanced collateral conductance in dogs with progressive single-vessel coronary occlusion. The beneficial effect of bFGF occurred primarily between the 7th and 14th days of therapy, and regression of collateral development was not noted after withdrawal of treatment. The present investigation provides impetus to the concept that collateral development can be enhanced pharmacologically-specifically by bFGF-raising the possibility that such an intervention might eventually be applied clinically.

PMID: 7805195 [PubMed - indexed for MEDLINE]

 
Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs.

Rajanayagam MA, Shou M, Thirumurti V, Lazarous DF, Quyyumi AA, Goncalves L, Stiber J, Epstein SE, Unger EF.

Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1650, USA.

OBJECTIVE: In preparation for clinical trials of basic fibroblast growth factor (bFGF) to treat ischemic heart disease, we sought to identify a clinically feasible method of bFGF administration. BACKGROUND: Basic FGF has been shown to promote collateral development after experimentally induced coronary occlusion; however, methods of bFGF delivery that have been shown to be effective in previous investigations would not be practical for clinical use. METHODS: Four randomized, blinded, controlled investigations were conducted independently and sequentially in an established canine model. For all studies, dogs underwent operative placement of proximal left circumflex coronary artery ameroid constrictors. The four investigational regimens included: 1) bFGF by central venous bolus injection, 1,740 microg/day for one, two or seven days; 2) bFGF by intravenous infusion, 100 microg/kg body weight per day for seven days; 3) bFGF by pericardial instillation, 2,000 microg/day for 7 days; and 4) bFGF by intracoronary injection (Judkin's technique), 100 microg/kg per day for one or two days. Each substudy included a contemporaneous vehicle control group. Collateral perfusion (microspheres) was assessed during maximal coronary vasodilation during the first month after ameroid placement. RESULTS: Maximal collateral perfusion in dogs that received intracoronary bFGF for two days exceeded that of concurrent control dogs by 31% (p < 0.01). Perfusion was not increased in dogs that received single-dose intracoronary bFGF. Basic FGF administration by central venous bolus injection, intravenous infusion and pericardial injection failed to enhance collateral perfusion. CONCLUSIONS: Administration of bFGF by the intracoronary route, an intervention that is feasible in patients, augments collateral development in dogs. These data provide a rationale for clinical testing of intracoronary bFGF in ischemic heart disease.

PMID: 10676703 [PubMed - indexed for MEDLINE]

 
Basic fibroblast growth factor in a porcine model of chronic myocardial ischemia: a comparison of angiographic, echocardiographic and coronary flow parameters.

Lopez JJ, Edelman ER, Stamler A, Hibberd MG, Prasad P, Caputo RP, Carrozza JP, Douglas PS, Sellke FW, Simons M.

The Angiogenesis Research Center, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215, USA.

Recently, a number of growth factors including basic fibroblast growth factor (bFGF) have been shown to promote angiogenesis in vivo. In this study, we evaluated dose-dependent effect of bFGF administration in the setting of chronic myocardial ischemia. A total of 18 Yorkshire pigs subjected to ameroid occluder placement on the left circumflex artery were randomized to treatment with 10 (n = 6) or 100 microg (n = 5) of bFGF incorporated into heparin-alginate microspheres or inactive control pellets (n = 7). Eight weeks later, all animals underwent angiographic evaluation of collateral development as well as studies of coronary flow and global and regional left ventricular function. Both bFGF groups had significantly higher angiographic collateral index, TIMI flow scores and coronary flow in the ameroid-compromised territory compared with controls. Left ventricular function studies demonstrated improved global and regional function in both fibroblast growth factor groups with significantly better preservation of regional wall motion in high dose (100 microg) bFGF animals. We conclude that local perivascular delivery of bFGF results in significant improvement in myocardial function in the setting of chronic myocardial ischemia.

PMID: 9223578 [PubMed - indexed for MEDLINE]

 
Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia.

Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM.

Departments of Medicine, Biomedical Research, Surgery, and Anesthesiology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass 02135, USA.

BACKGROUND: We initiated a phase 1 clinical study to determine the safety and bioactivity of direct myocardial gene transfer of vascular endothelial growth factor (VEGF) as sole therapy for patients with symptomatic myocardial ischemia. METHODS AND RESULTS: VEGF gene transfer (GTx) was performed in 5 patients (all male, ages 53 to 71) who had failed conventional therapy; these men had angina (determined by angiographically documented coronary artery disease). Naked plasmid DNA encoding VEGF (phVEGF165) was injected directly into the ischemic myocardium via a mini left anterior thoracotomy. Injections caused no changes in heart rate (pre-GTx=75+/-15/min versus post-GTx=80+/-16/min, P=NS), systolic BP (114+/-7 versus 118+/-7 mm Hg, P=NS), or diastolic BP (57+/-2 versus 59+/-2 mm Hg, P=NS). Ventricular arrhythmias were limited to single unifocal premature beats at the moment of injection. Serial ECGs showed no evidence of new myocardial infarction in any patient. Intraoperative blood loss was 0 to 50 cm3, and total chest tube drainage was 110 to 395 cm3. Postoperative cardiac output fell transiently but increased within 24 hours (preanesthesia=4.8+/-0.4 versus postanesthesia=4.1+/-0.3 versus 24 hours postoperative=6. 3+/-0.8, P=0.02). Time to extubation after closure was 18.4+/-1.4 minutes; average postoperative hospital stay was 3.8 days. All patients had significant reduction in angina (nitroglycerin [NTG] use=53.9+/-10.0/wk pre-GTx versus 9.8+/-6.9/wk post-GTx, P<0.03). Postoperative left ventricular ejection fraction (LVEF) was either unchanged (n=3) or improved (n=2, mean increase in LVEF=5%). Objective evidence of reduced ischemia was documented using dobutamine single photon emission computed tomography (SPECT)-sestamibi imaging in all patients. Coronary angiography showed improved Rentrop score in 5 of 5 patients. CONCLUSIONS: This initial experience with naked gene transfer as sole therapy for myocardial ischemia suggests that direct myocardial injection of naked plasmid DNA, via a minimally invasive chest wall incision, is safe and may lead to reduced symptoms and improved myocardial perfusion in selected patients with chronic myocardial ischemia.

Publication Types:

• Clinical Trial
• Clinical Trial, Phase I

PMID: 9860779 [PubMed - indexed for MEDLINE]

 
Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease.

Symes JF, Losordo DW, Vale PR, Lathi KG, Esakof DD, Mayskiy M, Isner JM.

Department of Surgery, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA. jsymes@semc.org

BACKGROUND: Patients presenting with medically intractable angina who have undergone previous coronary bypass (CABG) and/or percutaneous revascularization procedures are frequently deemed "inoperable" based on angiographic findings of diffuse distal disease or a lack of available conduits. We initiated a phase I clinical trial to assess the safety and bioactivity of intramyocardial transfection of plasmid DNA encoding for the angiogenic mitogen vascular endothelial growth factor (ph-VEGF165) in such patients. METHODS: phVEGF165 (125 microg, n = 10; 250 microg, n = 10) was injected directly into the myocardium through a mini left anterior thoracotomy as sole therapy in 20 patients (15 male, 5 female, age 48 to 74 years) with class III or IV angina, reversible ischemia on stress sestamibi scans, and "inoperable" coronary artery disease. RESULTS: All patients tolerated surgery uneventfully and were extubated on the table. No perioperative myocardial infarction, hemodynamic instability, or change in ventricular function occurred. Mean hospital stay was 3.9 days. There was one late death (4 months). Plasma VEGF protein level increased from 30.6+/-4.1 pg/mL pretreatment to 73.7+/-10.1 pg/mL 14 days posttreatment (p = 0.0002) and returned to baseline by day 90. All 16 patients followed to day 90 reported a reduction in angina (nitroglycerin use/week = 60.2+/-4.9 preop vs 3.5+/-1.6 at 90 days; p<0.0001). Seventy percent (7 of 10) patients were completely angina free at 6 months. A reduction in ischemic defects on single photon emission computerized tomography sestamibi scans was observed in 13 of 17 patients at 60 days (7 of 8 in the 250-microg group). Stress perfusion score decreased from 19.4+/-3.7 at baseline to 15.9+/-3.4 at 60 days (p = 0.025). Angiographic evidence of improved collateral filling of at least one occluded vessel was observed in all patients evaluated at day 60. CONCLUSIONS: Direct myocardial gene transfer with phVEGF165 via a mini-thoracotomy can be performed safely and may result in significant symptomatic improvement in patients with "inoperable" coronary artery disease.

Publication Types:

• Clinical Trial
• Clinical Trial, Phase I

PMID: 10509970 [PubMed - indexed for MEDLINE]

 
Intraoperative multiplane transesophageal echocardiography for guiding direct myocardial gene transfer of vascular endothelial growth factor in patients with refractory angina pectoris.

Esakof DD, Maysky M, Losordo DW, Vale PR, Lathi K, Pastore JO, Symes JF, Isner JM.

Department of Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135, USA. esakof@pol.net

Gene transfer for therapeutic angiogenesis represents a novel treatment for patients with chronic angina refractory to standard medical therapy and not amenable to conventional revascularization. We sought to assess the role of intraoperative multiplane transesophageal echocardiography (MPTEE) in guiding injection of naked DNA encoding vascular endothelial growth factor (VEGF) into the left ventricular (LV) myocardium of patients with refractory angina. After exposing the LV myocardium via a limited lateral thoracotomy, each of 17 patients in this series received 4 separate injections of VEGF DNA into different myocardial sites. Initial injections in the first patient produced intracavitary microbubbles, indicating injection of DNA into the LV chamber. Subsequently, each injection was preceded by a test injection of agitated saline. The absence of microbubbles while visualizing the LV cavity during the test injection verified that the ensuing injection of DNA would not be inadvertently squandered in the LV chamber itself. Intracavitary LV microbubbles were observed by MPTEE in 13 of 64 (20.3%) saline test injections and in 8 of 16 (50.0%) patients in which saline test injection was used, leading to adjustments in needle position. MPTEE imaging detected a previously unknown large, apical left ventricular thrombus in one patient, thereby preventing inadvertent injection of VEGF DNA through the myocardium into the thrombus. Imaging during and after injection verified no deleterious impact on LV function. We conclude that MPTEE is a useful tool for ensuring that myocardial gene therapy performed by direct needle injection results in gene transfer to the LV myocardium.

Publication Types:

• Clinical Trial

PMID: 10515450 [PubMed - indexed for MEDLINE]

 
Mechanism of impaired myocardial function during progressive coronary stenosis in conscious pigs. Hibernation versus stunning?

Shen YT, Vatner SF.

Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Mass.

The major goal of this study was to determine whether impaired myocardial contractile function during the development of progressive coronary artery stenosis induced by ameroid constriction in conscious pigs reflected myocardial "hibernation" or "stunning." Minipigs were instrumented with a coronary ameroid constrictor and hydraulic occluder, regional wall thickness crystals, a left ventricular (LV) pressure gauge, and aortic and left atrial catheters. In the seven pigs in which it was measured, systolic wall thickening (WT) distal to the ameroid fell by a maximum of 56 +/- 6% at 20 +/- 3 days after ameroid implantation and then began to recover. At 1 day after ameroid implantation, brief complete coronary artery occlusion (CAO) resulted in wall thinning distal to the ameroid (-113 +/- 4%) and transmural decreases in myocardial blood flow in endocardial (from 0.82 +/- 0.08 to 0.02 +/- 0.01 mL/min per gram) and epicardial (from 0.73 +/- 0.13 to 0.03 +/- 0.02 mL/min per gram) layers. At 20 +/- 3 days, baseline myocardial blood flow was not altered either in endocardial (0.92 +/- 0.10 mL/min per gram) or epicardial (0.85 +/- 0.12 mL/min per gram) layers, whereas brief complete coronary artery occlusion still reduced WT (-83 +/- 12%) and myocardial blood flow in endocardial (to 0.21 +/- 0.03 mL/min per gram) and epicardial (to 0.43 +/- 0.12 mL/min per gram) layers, indicating that the coronary artery was not totally occluded. Pathology in four pigs demonstrated no gross necrotic myocardium shortly after this time point. Transient reductions in WT distal to the ameroid were observed during progressive coronary artery stenosis in response to spontaneous increases in activity. Beat-by-beat analysis of these episodes revealed that acute reductions in WT followed increases in LV dP/dt and heart rate and exhibited delayed recovery. These data suggest that the reduced function during ameroid-induced coronary stenosis reflected cumulative myocardial stunning rather than a primary deficit in coronary blood flow or "hibernating myocardium."

PMID: 7859393 [PubMed - indexed for MEDLINE]

 
Hibernating myocardium.

Wijns W, Vatner SF, Camici PG.

Cardiovascular Center, Onze Lieve Vrouw Ziekenhuis, Aalst, Belgium.

Publication Types:

• Review
• Review, Tutorial

PMID: 9664095 [PubMed - indexed for MEDLINE]

48. Vale PR, Losordo DW, Milliken CE, et al. Randomized, placebo-controlled clinical study of percutaneous catheter-based left ventricular endocardial gene transfer of VEGF-2 for myocardial angiogenesis in patients with chronic myocardial ischemia [abstract]. Circulation. 2000;102:II-563.
 

 
Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery: results of a phase I randomized, double-blind, placebo-controlled trial.

Laham RJ, Sellke FW, Edelman ER, Pearlman JD, Ware JA, Brown DL, Gold JP, Simons M.

Angiogenesis Research Center and Interventional Cardiology Section, harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Mass., USA.

BACKGROUND:Angiogenesis is a promising treatment strategy for patients who are not candidates for standard revascularization, because it promotes the growth of new blood vessels in ischemic myocardium. METHODS AND RESULTS:We conducted a randomized, double-blind, placebo-controlled study of basic fibroblast growth factor (bFGF; 10 or 100 microg versus placebo) delivered via sustained-release heparin-alginate microcapsules implanted in ischemic and viable but ungraftable myocardial territories in patients undergoing CABG. Twenty-four patients were randomized to 10 microg of bFGF (n=8), 100 microg of bFGF (n=8), or placebo (n=8), in addition to undergoing CABG. There were 2 operative deaths and 3 Q-wave myocardial infarctions. There were no treatment-related adverse events, and there was no rise in serum bFGF levels. Clinical follow-up was available for all patients (16.0+/-6.8 months). Three control patients had recurrent angina, 2 of whom required repeat revascularization. One patient in the 10-microg bFGF group had angina, whereas all patients in the 100-microg bFGF group remained angina-free. Stress nuclear perfusion imaging at baseline and 3 months after CABG showed a trend toward worsening of the defect size in the placebo group (20.7+/-3.7% to 23.8+/-5.7%, P=0.06), no significant change in the 10-microg bFGF group, and significant improvement in the 100-microg bFGF group (19.2+/-5.0% to 9.1+/-5.9%, P=0.01). Magnetic resonance assessment of the target ischemic zone in a subset of patients showed a trend toward a reduction in the target ischemic area in the 100-microg bFGF group (10.7+/-3.9% to 3. 7+/-6.3%, P=0.06). CONCLUSIONS:This study of bFGF in patients undergoing CABG demonstrates the safety and feasibility of this mode of therapy in patients with viable myocardium that cannot be adequately revascularized.

Publication Types:

• Clinical Trial
• Clinical Trial, Phase I
• Randomized Controlled Trial

PMID: 10545430 [PubMed - indexed for MEDLINE]

 
Accelerated restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene transfer.

Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM.

Department of Medicine (Cardiology), St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass 02135, USA.

BACKGROUND: Delinquent reendothelialization (rET) has been shown to have a permissive, if not facilitatory, impact on smooth muscle cell proliferation. This inverse relation has been attributed to certain functions of the endothelium, including barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules. Accordingly, the present investigation was designed to test the hypothesis that an endothelial cell (EC) mitogen could serve as the basis for a novel gene therapy strategy designed to facilitate EC regeneration, reduce neointimal thickening, and promote recovery of EC dysfunction after balloon injury. METHODS AND RESULTS: New Zealand White rabbits underwent simultaneous balloon injury and gene transfer of one femoral artery with phVEGF165, encoding the 165-amino acid isoform of vascular endothelial growth factor (VEGF), or pGSVLacZ. In each animal transfected with phVEGF165 or pGSVLacZ, the contralateral femoral artery was also subjected to balloon injury but not to gene transfer. For pGSVLacZ, rET remained incomplete at 4 weeks after transfection; in contrast, phVEGF165 produced prompt rET, which was 95% complete by 1 week. Furthermore, rET in the contralateral, balloon-injured, nontransfected limb of the VEGF group was similarly accelerated. Consequently, intimal thickening was diminished, thrombotic occlusion was less frequent, and recovery of EC-dependent vasomotor reactivity was accelerated in VEGF transfectants compared with control animals. A similar benefit was observed for the contralateral, balloon-injured, nontransfected limb. CONCLUSIONS: Catheter-mediated, site-specific arterial gene transfer of phVEGF165 can accelerate rET at local and remote sites, leading to inhibition of neointimal thickening, reduction in thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity. These findings support the notion that gene transfer encoding for an EC-specific mitogen may be useful for preventing the complications, including restenosis, of balloon angioplasty.

PMID: 8989143 [PubMed - indexed for MEDLINE]

 
Passivation of metallic stents after arterial gene transfer of phVEGF165 inhibits thrombus formation and intimal thickening.

Van Belle E, Tio FO, Chen D, Maillard L, Chen D, Kearney M, Isner JM.

Department of Medicine, (Cardiology), St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

OBJECTIVES: This study sought to test the hypothesis that direct gene transfer of an endothelial cell mitogen could passivate metallic stents by accelerating endothelialization of the prosthesis. BACKGROUND: Thrombosis and restenosis comprise the principal clinical manifestations of compromised biocompatibility of endovascular stents. Previous studies have demonstrated that endothelial recovery at sites of balloon injury is a critical determinant of consequent intimal thickening and mural thrombus. We therefore investigated the potential for an endothelial cell mitogen delivered as plasmid DNA to optimize stent biocompatibility. METHODS: Naked plasmid DNA encoding vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) (phVEGF165) was delivered locally using a hydrogel-coated balloon angioplasty catheter to 16 rabbit iliac arteries in which metallic stents had been placed at the site of balloon injury; the contralateral iliac artery of each rabbit was balloon injured and stented but not transfected. RESULTS: Stent endothelialization was accelerated by phVEGF165 gene transfer (87.38 +/- 5.06% vs. 33.13 +/- 9.73% [mean +/- SEM] of the planimetered stent surface in the treated vs. contralateral limb, p = 0.005). This was associated with a significant reduction in mural thrombus (3.7 +/- 2.4% vs. 32.7 +/- 9.7%, p = 0.01) at day 7 and intimal thickening (maximal intimal area 0.61 +/- 0.09 vs. 1.44 +/- 0.12 mm2, p < 0.0001) at day 28. No benefit was observed from pCMV-luciferase in 14 similarly instrumented control rabbits. CONCLUSIONS: These findings indicate that arterial gene transfer of naked plasmid DNA encoding for an endothelial cell mitogen may successfully passivate endovascular stents by accelerating stent endothelialization, thereby reducing in-stent thrombus and obstruction due to intimal thickening.

PMID: 9137238 [PubMed - indexed for MEDLINE]

 
VEGF gene delivery to muscle: potential role for vasculogenesis in adults.

Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM.

Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.

Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures. Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues. The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels. Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels. No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected. These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent. Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.

PMID: 9844628 [PubMed - indexed for MEDLINE]

 
Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat--angiogenesis and angioma formation.

Schwarz ER, Speakman MT, Patterson M, Hale SS, Isner JM, Kedes LH, Kloner RA.

Heart Institute Research, Good Samaritan Hospital and Division of Cardiology, University of Southern California, Los Angeles 90017-2395, USA.

OBJECTIVES: The effects of direct intramyocardial injection of the plasmid encoding vascular endothelial growth factor (phVEGF165) in the border zone of myocardial infarct tissue in rat hearts were investigated. BACKGROUND: Controversy exists concerning the ability of VEGF to induce angiogenesis and enhance coronary flow in the myocardium. METHODS: Sprague-Dawley rats received a ligation of the left coronary artery to induce myocardial infarction (MI). At 33.1 +/- 6.5 days, the rats were injected with phVEGF165 at one location and control plasmid at a second location (500 microg DNA, n = 24) or saline (n = 16). After 33.1 +/- 5.7 days, the hearts were excised for macroscopic and histologic analysis. Regional blood flow ratios were measured in 18 rats by radioactive microspheres. RESULTS: phVEGF165-treated sites showed macroscopic angioma-like structures at the injection site while control DNA and saline injection sites did not. By histology, 21/24 phVEGF165-treated hearts showed increased focal epicardial blood vessel density and angioma-like formation. Quantitative morphometric evaluation in 20 phVEGF165-treated hearts revealed 44.4 +/- 10.5 vascular structures per field in phVEGF165-treated hearts versus 21.4 +/- 4.7 in control DNA injection sites (p < 0.05). Regional myocardial blood flow ratios between the injection site and noninfarcted area did not demonstrate any difference between phVEGF,165-treated hearts (0.9 +/- 0.2) and saline-treated hearts (0.7 +/- 0.1). CONCLUSIONS: Injection of DNA for VEGF in the border zone of MI in rat hearts induced angiogenesis. Angioma formation at the injection sites did not appear to contribute to regional myocardial blood flow, which may be a limitation of gene therapy for this application.

PMID: 10758976 [PubMed - indexed for MEDLINE]

 
Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1.

Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM.

Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0452, USA. gavint@itsa.ucsf.edu

Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) are endothelial cell-specific growth factors. Direct comparison of transgenic mice overexpressing these factors in the skin revealed that the VEGF-induced blood vessels were leaky, whereas those induced by Ang1 were nonleaky. Moreover, vessels in Ang1-overexpressing mice were resistant to leaks caused by inflammatory agents. Coexpression of Ang1 and VEGF had an additive effect on angiogenesis but resulted in leakage-resistant vessels typical of Ang1. Ang1 therefore may be useful for reducing microvascular leakage in diseases in which the leakage results from chronic inflammation or elevated VEGF and, in combination with VEGF, for promoting growth of nonleaky vessels.

PMID: 10617467 [PubMed - indexed for MEDLINE]

 
Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium.

Horowitz JR, Rivard A, van der Zee R, Hariawala M, Sheriff DD, Esakof DD, Chaudhry GM, Symes JF, Isner JM.

Department of Medicine (Cardiology), St Elizabeth's Medical Center, Tufts University School of Medicine, MA, USA.

In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 micrograms to anesthetized (n = 6) or conscious (n = 5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n = 7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 micrograms intravenous (IV; n = 5) or intracoronary (IC; n = 5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5 +/- 1.4% (P < .05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with N omega-nitro-L-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5 +/- 2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.

PMID: 9409257 [PubMed - indexed for MEDLINE]

 
VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts.

Hariawala MD, Horowitz JR, Esakof D, Sheriff DD, Walter DH, Keyt B, Isner JM, Symes JF.

Division of Cardiothoracic Surgery, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

Several recent studies have demonstrated the potential for improving myocardial perfusion by the continuous administration of angiogenic growth factors. Studies in our laboratory have shown that a single intraarterial or intravenous bolus of the endothelial cell specific mitogen vascular endothelial growth factor (VEGF) can significantly improve perfusion in a rabbit ischemic limb model. To test the efficacy of this therapeutic approach in chronic myocardial ischemia, 18 Yorkshire pigs underwent a left thoracotomy followed by placement of an ameroid constrictor around the proximal circumflex coronary artery. Gradual occlusion of the artery (26 +/- 4 days) was accompanied by identifiable hypokinesis of the posterolateral wall of the left ventricle (2D echo). Thirty days postoperatively, rhVEGF(165) (2 mg; n = 8) or saline (n = 10) was administered directly into the left coronary ostium. Postadenosine myocardial perfusion studies using colored microspheres 30 days later demonstrated superior blood flow in the ischemic zone of the VEGF-treated hearts (ischemic/normal ratio 1.09 vs 0.97, P < 0.05) compared with those receiving saline injection. Four of eight VEGF-treated animals succumbed, however, to severe hypotension following VEGF administration. Therefore 500 micrograms of VEGF were administered intracoronary to five normal pigs. A significant drop in mean arterial pressure (-44.4 +/- 3.2%, P < 0.05 vs baseline) and peripheral resistance (-13.2 +/- 4.5%, P < 0.05 vs baseline) was accompanied by increased heart rate. IV administration of N(omega)-nitro-L-arginine (L-NNA), an EDRF inhibitor, restored blood pressure to baseline. We conclude that a single intracoronary bolus of VEGF is capable of significantly augmenting flow to collateral-dependent ischemic myocardium. The associated hypotension appears to be EDRF-mediated. Further studies are needed to define the best dose and route of administration of VEGF for the treatment of coronary insufficiency.

PMID: 8661176 [PubMed - indexed for MEDLINE]

 
Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium.

van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM.

Department of Biomedical Research, St Elizabeth's Medical Center, Tufts University, School of Medicine, Boston, Mass 02135-2997, USA.

BACKGROUND: Vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) is an endothelial cell (EC) mitogen. This feature is considered central to the documented role of VEGF/VPF in promoting angiogenesis. More recent evidence suggests that VEGF/VPF may also serve a "maintenance" function, modulating various aspects of EC biology. In the present study, we sought to determine the extent to which VEGF/VPF may stimulate the release of NO from normal ECs. METHODS AND RESULTS: VEGF/VPF produced a dose-dependent rise in NO concentration ([NO]) from vascular segments of rabbit thoracic aorta, pulmonary artery, and inferior vena cava. In comparison to stimulation with acetylcholine, the onset of increased [NO] after administration of VEGF/VPF was slower, reaching a maximum value after 8 minutes. Preincubation of the aortic segments with L-arginine raised by twofold both baseline [NO] and [NO] stimulated by addition of 2.5 micrograms/mL VEGF/VPF. Removal of CaCl2 from the Krebs solution, disruption of the endothelium, and administration of NG-monomethyl-L-arginine abrogated the stimulatory effect of 10 micrograms/mL VEGF/VPF. Similar findings were documented with an NO-specific polarographic electrode to measure NO released from cultured human umbilical vein ECs. CONCLUSIONS: VEGF/VPF stimulates production of NO from rabbit and human ECs. This finding (1) constitutes inferential evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit as well as human ECs and (2) supports the notion that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of EC NO.

PMID: 9054767 [PubMed - indexed for MEDLINE]

 
Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, Huang PL, Isner JM.

Department of Medicine (Cardiology) and Department of Cardiothoracic Surgery and Biomedical Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

PMID: 9616228 [PubMed - indexed for MEDLINE]

63. Vale PR, Wuensch DI, Rauh GF, Rosenfield K, Schainfeld RM, Isner JM. Arterial gene therapy for inhibiting restenosis in patients with claudication undergoing superficial femoral artery angioplasty. Circulation. 1998;98:I-66.

 
Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty.

Laitinen M, Hartikainen J, Hiltunen MO, Eranen J, Kiviniemi M, Narvanen O, Makinen K, Manninen H, Syvanne M, Martin JF, Laakso M, Yla-Herttuala S.

A.I. Virtanen Institute, University of Kuopio, Finland.

Blood vessels are among the easiest targets for gene therapy. However, no data are available about the safety and feasibility of intracoronary gene transfer in humans. We studied the safety and efficacy of catheter-mediated vascular endothelial growth factor (VEGF) plasmid/liposome (P/L) gene transfer in human coronary arteries after percutaneous translumenal coronary angioplasty (PTCA) in a randomized, double-blinded, placebo-controlled study. The optimized angioplasty/gene delivery method was previously shown to lead to detectable VEGF gene expression in human peripheral arteries as analyzed from amputated leg samples. Gene transfer to coronary arteries was done with a perfusion-infusion catheter, using 1000 microg of VEGF or beta-galactosidase plasmid complexed with 1000 microl of DOTMA:DOPE liposomes. Ten patients received VEGF P/L, three patients received beta-galactosidase P/L, and two patients received Ringer lactate. Gene transfer to coronary arteries was feasible and well tolerated. Except for a slight increase in serum C-reative protein in all study groups, no adverse effects or abnormalities in laboratory parameters were detected. No VEGF plasmid or recombinant VEGF protein was present in the systemic circulation after the gene transfer. In control angiography 6 months later, no differences were detected in the degree of coronary stenosis between treatment and control groups. We conclude that catheter-mediated intracoronary gene transfer performed after angioplasty is safe and well tolerated and potentially applicable for the prevention of restenosis and myocardial ischemia.

Publication Types:

• Clinical Trial
• Randomized Controlled Trial

PMID: 10680840 [PubMed - indexed for MEDLINE]