Ann Pieczek

Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb

BACKGROUND Preclinical findings suggest that intra-arterial gene transfer of a plasmid which encodes for vascular endothelial growth factor (VEGF) can improve blood supply to the ischaemic limb. We have used the method in a patient. METHODS Our patient was the eighth in a dose-ranging series. She was aged 71 with an ischaemic right leg. We administered 2,000 micrograms human plasmid phVEGF165 that was applied to the hydrogel polymer coating of an angioplasty balloon. By inflating the balloon, plasmid DNA was transferred to the distal popliteal artery. FINDINGS Digital subtraction angiography 4 weeks after gene therapy showed an increase in collateral vessels at the knee, mid-tibial, and ankle levels, which persisted at a 12-week view. Intra-arterial doppler-flow studies showed increased resting and maximum flows (by 82% and 72%, respectively). Three spider angiomas developed on the right foot/ankle about a week after gene transfer; one lesion was excised and revealed proliferative endothelium, the other two regressed. The patient developed oedema in her right leg, which was treated successfully. INTERPRETATION Administration of endothelial cell mitogens promotes angiogenesis in patients with limb ischaemia.

Vascular Endothelial Growth Factor165 Gene Transfer Augments Circulating Endothelial Progenitor Cells in Human Subjects

Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%, P<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector, P<0.001; VEGF versus saline, P<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, alpha(v)beta(3), and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.

Histopathology of In-Stent Restenosis in Patients With Peripheral Artery Disease

BACKGROUND Clinical studies have suggested that smooth muscle cell (SMC) hyperplasia is the most likely cause of in-stent restenosis. However, pathological data regarding this issue are limited. Specifically, direct evidence of proliferative activity in tissues excised from stenotic stents has not been previously reported. METHODS AND RESULTS Tissue specimens were retrieved by directional atherectomy from 10 patients in whom in-stent restenosis complicated percutaneous revascularization of peripheral artery disease. Analysis of cellular composition was performed quantitatively after cell-specific immunostaining. For specimens preserved in methanol (7 of 10), cellular proliferation was evaluated by use of antibodies to proliferating cell nuclear antigen (PCNA), cyclin E, and cdk2. TUNEL staining for apoptosis was performed on 8 paraformaldehyde-preserved specimens. Each of the 10 specimens contained extensive foci of hypercellularity composed predominantly of SMCs (mean+/-SEM, 59.3+/-3.0%). Evidence of ongoing proliferative activity was documented in all 7 methanol-preserved specimens: 24.6+/-2.3% of SMCs were PCNA-positive, 24.8+/-3.1% were cyclin E-positive, and 22.5+/-2.2% were cdk2-positive. Apoptotic cells were detected in all 8 specimens that had been appropriately preserved to permit DNA nick-end labeling. Macrophages and leukocytes were identified in each of the 10 specimens but accounted for a proportionately smaller number of cells (14.5+/-1.9% and 9.5+/-1.4%, respectively). Organized thrombus was observed in 6 of the 10 specimens. CONCLUSIONS These findings support the notion that in-stent restenosis results from SMC hyperplasia and suggest that adjunctive therapies designed to inhibit SMC proliferation may further enhance the utility of endovascular stents.

Focal compensatory enlargement of human arteries in response to progressive atherosclerosis. In vivo documentation using intravascular ultrasound.

BACKGROUND Previous postmortem studies have demonstrated compensatory enlargement of atherosclerotic arteries in animal models and patients. Conclusions regarding these changes were drawn based on a comparison of the dimensions of diseased arteries in one group of subjects with the dimensions of normal arteries in another group. This method admits potential confounding variables, such as demographics and other disease states, which might also have an impact on arterial size. METHODS AND RESULTS Using intravascular ultrasound, we studied a total of 62 paired, adjacent normal and diseased sites in the superficial femoral arteries of 20 patients undergoing peripheral vascular interventions. Morphological assessment was performed using a computer-based image analysis system. Measurements were made of the cross-sectional area of the arterial lumen, the atherosclerotic plaque, and the outer border of the artery. These dimensions were then compared to determine the effects of progressive atherosclerosis on arterial morphology. Luminal cross-sectional area decreased from 21.1 +/- 2.2 mm2 in normal segments to 16.7 +/- 0.8 mm2 (P = .0001) in adjacent atherosclerotic segments. Similarly, minimal luminal diameter decreased from 5.7 +/- 0.2 to 5.0 +/- 0.1 mm2, and maximal luminal diameter decreased from 6.2 +/- 0.2 to 5.7 +/- 0.2 mm2. At these same sites, total arterial area was 32.9 +/- 1.6 and 37.9 +/- 1.9 mm2 (P = .0001) in normal and diseased segments, respectively. Minimal and maximal arterial diameters demonstrated similar increases (7.3 +/- 0.2 to 7.7 +/- 0.2 mm2 [P = .0015] and 7.6 +/- 0.2 to 8.3 +/- 0.2 mm2 [P = .0001], respectively). Regression analysis disclosed correlation of the cross-sectional area of plaque to the total arterial area (R = .70, P = .0001). CONCLUSIONS Human arteries enlarge in response to progressive atherosclerosis. This compensatory mechanism results in an increase in arterial size that is proportionate to the cross-sectional area of plaque that has accumulated in the vessel. Intravascular ultrasound demonstrates that this process is focal compensatory enlargement at discrete sites of atherosclerotic narrowing immediately adjacent to more normal areas in which arterial size is smaller.

Arterial Gene Therapy for Therapeutic Angiogenesis in Patients With Peripheral Artery Disease

### Peripheral Artery Disease: Primary Pharmacological Therapy Is Ineffective for Patients With Critical Limb Ischemia The prognosis for patients with chronic critical leg ischemia, ie, rest pain and/or established lesions that jeopardize the integrity of the lower limbs, is often poor. Psychological testing of such patients has typically disclosed quality-of-life indexes similar to those of patients with cancer in critical or even terminal phases of their illness.1 It has been estimated that in toto,2 150 000 patients per year require lower-limb amputations for ischemic disease in the United States. Their prognosis after amputation is even worse3 : the perioperative mortality for below-knee amputation in most series is 5% to 10% and for above-knee amputation 15% to 20%. Even when they survive, nearly 40% will have died within 2 years of their first major amputation; a major amputation is required in 30% of cases; and full mobility is achieved in only 50% of below-knee and 25% of above-knee amputees. These grim statistics are compounded by the lack of efficacious drug therapy. As concluded in the Consensus Document of the European Working Group on Critical Leg Ischemia,3 “. . .there presently is inadequate evidence from published studies to support the routine use of primary pharmacological treatment in patients with critical leg ischemia. . . .” Evidence for the utility of medical therapy in the treatment of claudication is no better.4 5 Consequently, the need for alternative treatment strategies in such patients is compelling. ### Therapeutic Angiogenesis Is a Novel Strategy for the Treatment of Critical Limb Ischemia The therapeutic implications of angiogenic growth factors were identified by the pioneering work of Folkman6 and other workers more than two decades ago. Beginning a little more than a decade ago,7 a series of polypeptide growth factors were purified, sequenced, and demonstrated to be responsible for natural as well as pathological angiogenesis. More recent investigations have established the feasibility of using recombinant formulations of such angiogenic growth …

Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) was discovered as a tumor-secreted factor that augments vascular permeability (1). The permeability-enhancing effect of VEGF has been estimated to be 50 times greater than that of histamine (2). After identification of the effects of VEGF on microvascular permeability, Leung (3) and Keck (4) and their colleagues demonstrated that VEGF may promote endothelial cell proliferation and migration. These findings led to preclinical animal studies (5-7) and human studies (8-10) that established that VEGF may promote angiogenesis in cases of limb ischemia. We prospectively evaluated 90 patients with peripheral artery disease who underwent gene transfer of naked plasmid DNA encoding the 165amino acid isoform of VEGF (phVEGF165) for clinical evidence of enhanced vascular permeability. The primary results of these trials have been reported in preliminary form elsewhere (9-12). Methods Between December 1994 and July 1999, 90 patients (mean age SD, 59 19 years) underwent gene transfer of phVEGF165 as therapeutic angiogenesis (13) or to prevent restenosis after angioplasty (14). The protocols for these trials were approved by the institutional review board and institutional biosafety committee of St. Elizabeths Medical Center, the Recombinant DNA Advisory Committee of the National Institutes of Health, and the Food and Drug Administration. Informed consent was obtained from all patients treated. Intra-arterial gene transfer was performed in 40 patients. Of these patients, 28 with claudication underwent phVEGF165 gene transfer after superficial femoral artery angioplasty; 4 patients with lower-extremity pain at rest and 8 patients with gangrene were treated to promote angiogenesis in the ischemic limb. Plasmid doses were 100 g (1 patient), 500 g (1 patient), 1000 g (13 patients), 2000 g (22 patients), and 4000 g (3 patients). Intramuscular gene transfer was performed in 50 patients, of whom 13 were treated for pain at rest and 37 presented with established gangrene. Five patients underwent treatment of the contralateral limb 3 or more months after treatment of the initial limb. Patients received 1000 g (n =10), 2000 g (n =19), 3000 g (n =11), and 4000 g (n =10) of phVEGF165. Edema was scored jointly by two observers as follows: 0, no edema; 1, edema limited to the foot; 2, edema involving the foot and ankle; 3, edema involving the calf; 4, more than the three preceding symptoms (Figure). Figure. Representative examples of lower-extremity edema ( asterisks ) according to clinical grade in four patients who underwent intramuscular gene transfer of naked plasmid DNA encoding vascular endothelial growth factor. Venous blood samples were analyzed by using enzyme-linked immunosorbent assay at baseline and weekly up to 4 weeks after the initial gene transfer, as described elsewhere (8). Data are reported as the mean (SE). The relation between nominal variables was calculated by using the Fisher exact test (2 2 contingency table). All statistical tests were two-tailed. A P value less than 0.05 indicated statistical significance. Results Development of Edema Transient lower-extremity edema was observed after gene transfer of phVEGF165 in 31 of 90 (34%) patients. Edema was graded as 1 in 9 patients, 2 in 13 patients, 3 in 6 patients, and 4 in 3 patients. In 3 of 90 (3.3%) patients, edema developed in both limbs after gene transfer of phVEGF165. All 3 patients had critical limb ischemia in both lower extremities. Relation of Edema to Tissue Integrity The incidence of peripheral edema differed among all subgroups. Peripheral edema developed significantly less frequently in patients without compromised tissue integrity than in patients with ischemic ulcers or gangrene. Peripheral edema associated with gene transfer was observed in 0 of 28 (0%) patients with claudication, 4 of 17 (24%) patients with pain at rest, and 27 of 45 (60%) patients with gangrene (Table). Results of the Fisher exact test showed that the incidence of edema differed significantly among all patient groups. Specifically, 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 was less common in patients with pain at rest than in those with ischemic ulcers (P =0.017). Table. Frequency of Peripheral Edema after Intra-Arterial and Intramuscular Administration of Naked Plasmid DNA Encoding Vascular Endothelial Growth Factor Intra-Arterial Compared with Intramuscular Gene Transfer Peripheral edema was observed after both intra-arterial and intramuscular gene transfer (Table). After intra-arterial gene transfer, peripheral edema occurred in 0 of 28 (0%) patients with claudication, 1 of 4 (25%) patients with pain at rest, and 4 of 8 (50%) patients with established gangrene. After intramuscular gene transfer, peripheral edema occurred in 3 of 13 (23%) patients with pain at rest and 23 of 37 (62%) patients with gangrene. The difference between the incidence of lower-extremity edema after intra-arterial gene transfer (5 of 40 patients [12.5%]) compared with intramuscular gene transfer (26 of 55 patients [52%]) was statistically significant (P <0.001) only when patients with claudication were included in the intra-arterial gene transfer group. When comparison of intra-arterial and intramuscular gene transfer was limited to patients in whom either method was used to treat pain at rest or gangrene, the incidence of edema did not differ significantly (5 of 12 patients [42%] compared with 26 of 50 patients [52%], respectively; P >0.2). Development of edema was unrelated to the dose of phVEGF165. Time to Development of Edema Clinically apparent peripheral edema usually developed within 3 weeks after intra-arterial or intramuscular gene transfer. Development of edema corresponded temporally to an increase in circulating levels of VEGF, consistent with the time course of gene expression (2 to 3 weeks) established for this plasmid in preclinical animal studies (7, 10). No differences were observed for intra-arterial compared with intramuscular gene transfer with regard to the time to development of edema or increased serum levels of VEGF. Despite the temporal relation between the increase in serum VEGF levels and development of peripheral edema, no correlation was observed between the absolute peak level of VEGF and the appearance of peripheral edema. Similarly, no relation was seen between the absolute or relative increase in serum VEGF levels from baseline to peak levels and the presence or absence of edema. Neither the magnitude (scores of 1 to 4) nor the probability of lower-extremity edema could be predicted by an individual VEGF level. Recurrent Edema, Treatment, and Hospitalization Development of peripheral edema was usually consistent among patients treated more than once. All patients who developed edema after the first gene transfer developed edema on subsequent injections, whereas no patient in whom edema failed to develop after the first gene transfer experienced edema on repeated gene transfer. Treatment was usually initiated on an outpatient basis and consisted of oral diuretics. Patients most commonly received furosemide, bumetanide, or hydrochlorothiazide or a combination of these agents. Edema was promptly attenuated after administration of diuretics and resolved completely within 2 to 4 weeks after initiation of therapy. In five patients, diuretics were administered intravenously during hospital admission; in none of these five patients was edema the principal indication for hospitalization. Four of the five patients were admitted for initiation of antibiotic treatment for suspected osteomyelitis, and one patient was admitted for control of ischemic pain at rest. Vascular Endothelial Growth FactorEnhanced Vascular Permeability and Evidence of Angiogenesis Evidence of augmented collateral vessel development, including an increase in the anklebrachial or toebrachial index to a value greater than 0.1, newly visible collateral vessels on follow-up serial angiography, or marked improvement of ischemic gangrene and disappearance of ischemic rest pain, was seen in 43 of the 57 patients treated for critical limb ischemia. The relation between development of edema and evidence of enhanced angiogenesis was not statistically significant (P >0.2). Discussion Our findings show that VEGF may augment vascular permeability in humans. The fact that edema failed to develop in patients with claudication but was observed in nearly half of the patients with resting ischemia suggests that the permeability-enhancing effects of VEGF are directly or indirectly potentiated by tissue ischemia. Ischemic damage to the integrity of the microcirculation may directly potentiate the effect of VEGF. Similar loss of microcirculatory integrity in patients with critical limb ischemia frequently leads to edema after conventional revascularization; in that setting, successful bypass surgery leads to augmented blood flow, which, when superimposed on a damaged endothelial substrate, allows excess fluid transudation and, subsequently, development of clinically apparent edema. The potent vasodilating effects of VEGF, previously shown to be a potent stimulus for release of nitric oxide (15), together with vasoactive metabolites released from ischemic tissues may compound this effect. These mechanisms may have contributed to the development of bilateral edema in three patients with critical limb ischemia involving both lower extremities. In the absence of ischemia at rest, however, even the potent permeability-enhancing effects of VEGF were insufficient to cause clinically apparent edema. Systemic administration of VEGF in a rabbit model of hind-limb ischemia was shown to cause selective neovascularization of the ischemic but not normally vascularized areas of the limb (5, 16). The localized biological effect observed in these experimental models, in which angiogenesis was selectively targeted to hy

Use of a Constitutively Active Hypoxia-Inducible Factor-1α Transgene as a Therapeutic Strategy in No-Option Critical Limb Ischemia Patients Phase I Dose-Escalation Experience

Background— Critical limb ischemia, a manifestation of severe peripheral atherosclerosis and compromised lower-extremity blood flow, results in a high rate of limb loss. We hypothesized that adenoviral delivery of a constitutively active form of the transcription factor hypoxia-inducible factor-1&agr; (ie, Ad2/HIF-1&agr;/VP16 or HIF-1&agr;) into the lower extremity of patients with critical limb ischemia would be safe and might result in a durable clinical response. Methods and Results— This phase I dose-escalation program included 2 studies: a randomized, double-blind, placebo-controlled study and an open-label extension study. In total, 34 no-option patients with critical limb ischemia received HIF-1&agr; at doses of 1×108 to 2×1011 viral particles. No serious adverse events were attributable to study treatment. Five deaths occurred: 3 in HIF-1&agr; and 2 in placebo patients. In the first (randomized) study, 7 of 21 HIF-1&agr; patients met treatment failure criteria and had major amputations. Three of the 7 placebo patients rolled over to receive HIF-1&agr; in the extension study. No amputations occurred in the 2 highest-dose groups of Ad2/HIF-1&agr;/VP16 (1×1011 and 2×1011 viral particles). The most common adverse events included peripheral edema, disease progression, and peripheral ischemia. At 1 year, limb status observations in HIF-1&agr; patients included complete rest pain resolution in 14 of 32 patients and complete ulcer healing in 5 of 18 patients. Conclusions— HIF-1&agr; therapy in patients with critical limb ischemia was well tolerated, supporting further, larger, randomized efficacy trials.

How does angioplasty work? Serial analysis of human iliac arteries using intravascular ultrasound.

BackgroundPrevious studies regarding the mechanism by which balloon angioplasty increases luminal patency have generally used animal models or postmortem specimens from occasional fatal cases of angioplasty performed in human patients. In either case, conclusions regarding participatory mecha-nisms have relied exclusively on nonserial, postangioplasty histopathological examination. Methods and ResultsIn the present study, intravascular ultrasound examination was performed before and after balloon angioplasty in 40 consecutive patients with iliac artery stenoses. The areas of the arterial wall, plaque, lumen, and areas resulting from angioplasty-induced plaque fractures were measured immediately after angioplasty in vivo and compared with findings recorded immediately before angio-plasty. Angioplasty increased luminal cross-sectional area (CSA) from 11.5 ± 0.6 mm2 before angioplasty to 25.4 ± 1.2 mm2 after angioplasty (p=0.0001). CSA of the portion of the postangioplasty neolumen contained within angioplasty-induced plaque fractures measured 10.0 ± 0.8 mm2; the neolumen excluding the area contributed by these plaque fractures measured 15.4 ± 0.8 mm2. Thus, the area contained within plaque fractures accounted for 10.0 mm2 (71.9%) of the 13.9-mm2 increase in luminal CSA after angioplasty. Analysis of CSA occupied by atherosclerotic plaque disclosed that plaque CSA decreased from 33.8 ± 1.8 mm2 before angioplasty to 22.5 ± 1.5 mm2 after angioplasty (p=0.0001). Plaque CSA was thus reduced (“compressed”) by 11.3 ± 1.1 mm2. Total artery CSA increased (“stretched”) slightly from 45.3 ± 2.6 mm2 before angioplasty to 47.8 ± 2.0 mm2 after angioplasty (p=0.0025). ConclusionsIn vivo analysis of iliac stenoses by intravascular ultrasound immediately before and after angioplasty demonstrates that plaque fractures and “compression” of atherosclerotic plaque are the principal factors responsible for increased luminal patency resulting from balloon angioplasty. “Stretch-ing” of the arterial wall provides an additional, but minor, contribution.

Restenosis of Endovascular Stents From Stent Compression

OBJECTIVES We sought to determine the basis for restenosis within superficial femoral arteries (SFAs) and hemodialysis conduits treated with balloon-expandable stents. BACKGROUND Use of stents within coronary and peripheral vessels continues to increase exponentially. The mechanism of restenosis within stents placed at various vascular sites is not well understood. In particular, the implications of deploying a balloon-expandable stent in a compressible site are not well understood. METHODS After the serendipitous detection of stent deformation during intravascular ultrasound (IVUS) examination of a restenosed dialysis fistula, we evaluated a consecutive series of patients with stents placed in compressible vascular sites, including the SFA (six patients) and hemodialysis fistulae (five patients). Clinical, angiographic and IVUS examinations were performed to evaluate mechanisms of restenosis. RESULTS Stent compression was identified as the principal cause of restenosis in all dialysis conduits and SFAs. Stent deformity was not reliably identified by angiography; however, IVUS identified compression of two forms: eccentric deformation, implicating two-point compressive force, and complete circumferential encroachment of stent struts around the catheter, suggesting multidirectional compressive force. Despite redilation, secondary restenosis resulting from recurrent compression recurred in most sites. CONCLUSIONS Restenosis within balloon-expandable endovascular stents may occur as a result of stent compression, a phenomenon readily detected by IVUS, but often not by angiography. These findings have significant implications for the use of balloon-expandable stents within vascular sites subject to extrinsic compression, such as hemodialysis conduits, the adductor canal segment of the SFA and carotid arteries.

Vascular Endothelial Growth Factor Gene Transfer for Diabetic Polyneuropathy: A Randomized, Double-Blinded Trial

Randomized, blinded trial of intramuscular gene transfer using plasmid vascular endothelial growth factor (VEGF) to treat diabetic polyneuropathy.