Yoram Vodovotz

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

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.

Effects of Intracoronary Radiation on Thrombosis After Balloon Overstretch Injury in the Porcine Model

BACKGROUND The main complications of PTCA remain thrombosis and restenosis. Recent studies have demonstrated reduction in the neointimal hyperplasia after intracoronary radiation (IR) with doses of 10 to 25 Gy of ionizing radiation delivered by either beta- or gamma-emitters to injured vessels. The purpose of this study was to examine the effect of ionizing radiation on the thrombosis rate (TR) of injured porcine coronary arteries. METHODS AND RESULTS Thirty-four juvenile swine (63 coronary arteries) were subjected to overstretch balloon injury followed by IR with doses of 0 to 18 Gy of either beta- or gamma-radiation. Two weeks after treatment, tissue sections were perfusion-fixed, stained with hematoxylin-eosin and Verhoeff-van Giesons stain, and analyzed for presence of a thrombus, thrombus morphology, and neointima formation by computer-assisted histomorphometry techniques. Although the overall TR increased dose-dependently from 0 to 18 Gy prescribed dose, luminal thrombi decreased. Thrombus area also decreased with increasing radiation dose, whether assessed at the prescription point or at the luminal surface, which corresponded to decreased intimal area. Furthermore, luminal thrombi present after IR tended to consist mostly of fibrin and thus were less organized than in controls. CONCLUSIONS These results suggest that IR induces thrombosis but does not necessarily compromise the lumen. Strategies for reducing TR may further decrease intimal area as well as increasing the safety of this therapy.

Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: role of TNF-alpha.

Macrophages are activated to become cytotoxic by a highly coordinated set of cytokine signals. Ionizing radiation can mimic cytokine signals and lead to enhanced states of activation. We tested the ability of γ‐radiation, alone and with interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS), to induce nitric oxide (NO) production in J774.1 and RAW264.7 murine macrophages. NO was induced weakly, moderately, or strongly by IFN‐γ alone, LPS alone, or IFN‐γ + LPS, respectively. Radiation alone (0.5–50 Gy) did not induce NO, but enhanced NO production in a dose‐dependent manner (0.5–5 Gy) when cells were exposed to IFN‐γ or LPS 24 h post‐irradiation. Immunoblots showed parallel induction of nitric oxide synthase (NOS2). Application of antitumor necrosis factor α (TNF‐α) antibody before irradiation blocked induction of NO by IFN‐γ. We conclude (1) that irradiated cells produce more NO in response to either IFN‐γ or LPS and (2) that the increase is mediated by induction of TNF‐α. J. Leukoc. Biol. 64: 459–466; 1998.

Intravascular radiation accelerates atherosclerotic lesion formation of hypercholesteremic rabbits.

OBJECTIVES The purpose of the present study is to evaluate the effect of intravascular radiation (IR) on the arterial wall of uninjured vessels in the hypercholesteremic rabbit model. METHODS Aortas of 24 New Zealand white rabbits were treated with either intravascular 192-Ir gamma-radiation (15 Gy at 2 mm from the center of the source) or were exposed to the source catheter without radiation (sham controls). Following the radiation treatment, the animals were fed a 2% cholesterol diet until euthanasia at 2 (n=8) or 6 (n=16) weeks. Arteries were analyzed using light and scanning electron microscopy (SEM); transforming growth factor beta (TGF-beta) 1, a promoter of connective tissue deposition, was also monitored. RESULTS At 2 weeks, SEM analysis showed well-aligned endothelial cells in nonradiated segments, whereas irradiated arteries consistently contained adherent and subendothelial macrophages with focal areas of endothelial disruption. Further radiated segments at 2 weeks showed a 7-fold increase in active TGF beta-1 over nonradiated segments. At 6 weeks, there was a significant increase in plaque and vessel wall area relative to control arteries, however, no differences were noted in the density of actin-positive smooth muscle cells (SMCs) or macrophages. Similarly, no differences were noted in cell proliferation between groups as evidenced by the marker bromodeoxyuridine (BrdU). In contrast, nonirradiated segments frequently contained cellular areas with extracellular lipid. CONCLUSION Exposure of previously uninjured vessels to IR and hypercholesterolemia is associated with increased plaque burden and leads to more advanced plaque types. Special care should be taken to minimize radiation exposure in normal vascular segments in hypercholesterolemic patients undergoing radiation therapy.

Laser Myocardial Revascularization Modulates Expression of Angiogenic, Neuronal, and Inflammatory Cytokines in a Porcine Model of Chronic Myocardial Ischemia

Abstract  Background: Controversy exists whether transmyocardial laser revascularization (TMR) is associated with angiogenesis or neuromodulation and whether these are time‐dependent phenomena. Accordingly, we performed a time‐course analysis of the expression of angiogenic and neuronal factors following experimental percutaneous TMR. Methods and Results: Five weeks after placing ameroid constrictors on the circumflex coronary artery, 16 pigs underwent left ventricular mapping guided TMR using Ho:YAG laser (2 J × 1 pulse) at 30 sites directed at the ischemic zones and 11 animals were ischemic controls. Histology and immunostaining were obtained at 1 and 2 weeks (4 TMR and 3 controls at each time point) and at 4 weeks (8 TMR and 5 controls) for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), nerve growth factor (βNGF), substance P (SP), and monocyte chemoattractant protein‐1 (MCP‐1). Immunoreactivity was scored using a digital image analysis system. Factor VIII staining was used for blood vessel counting. Enhanced regional expression of VEGF, bFGF and MCP‐1 in the TMR group was noted at 1 and 2 weeks with a threefold increase at 4 weeks following TMR compared to controls. βNGF expression in the TMR group was enhanced at 1 and 2 weeks with subsequent decline at 4 weeks to the controls level. SP expression was not significantly different between groups at all time points. There was a twofold increase in the number of blood vessels in the TMR group at 4 weeks, which was not apparent earlier. Conclusions: These immunohistological findings suggest that cytokines expression compatible with angiogenesis and neuromodulation occurs early after TMR. Up‐regulation of angiogenic and inflammatory cytokines may be more sustained than neuromodulation.

S-Nitrosoglutathione reduces nonocclusive thrombosis rate following balloon overstretch injury and intracoronary irradiation of porcine coronary arteries

PURPOSE Intracoronary radiation (IR) suppresses neointima formation following balloon injury in animal models. High doses of radiation exacerbate thrombosis and delay re-endothelialization. The free radical nitric oxide (NO) has been reported to inhibit platelet aggregation, reduce neointimal hyperplasia, and stimulate re-endothelialization. This study examined the effects of a chemical NO donor on neointima formation, thrombosis, and healing of irradiated porcine coronary arteries. METHODS AND MATERIALS Vascular lesions were created in the coronary arteries of 59 domestic swine by overstretch balloon injury. Arteries were then left untreated or were treated with intracoronary gamma-radiation using Iridium-192 in each artery to deliver 5 or 15 Gy at 2 mm from the center of the source. The chemical NO donor S-nitrosoglutathione (GSNO) was infused i.v. at a rate of 250 microg/min for 10 min before injury, followed by a continuous infusion for 60 min. Animals were euthanized at 14 days and their arteries were analyzed for histomorphometric indices of proliferation and thrombosis. RESULTS A dose of 15 Gy reduced the ratio of intimal area to medial fracture length (IA/FL) versus control (0.06 +/- 0.05 0.54 +/- 0.10 [p < 0. 001]) but increased the nonocclusive thrombosis rate compared to controls (85% vs. 30%; p < 0.05). A low dose of 5 Gy did not affect neointima formation. Treatment with GSNO reduced thrombosis in all treated groups: control, 15%; 5 Gy, 18%; and 15 Gy, 35% (p < 0.05) without affecting neointima formation. CONCLUSION Systemic administration of GSNO during balloon injury and IR was tolerated well by the swine and resulted in reduction of the thrombosis rate, especially at high doses, without apparent effect on neointima formation.

Differential remodeling after balloon overstretch injury and either β- or γ-intracoronary radiation of porcine coronary arteries

Abstract Background: Restenosis is a consequence of both neointimal hyperplasia and vessel remodeling. Prior studies have shown that intracoronary radiation (IR) prevents neointima accumulation, but its contribution to vessel remodeling is unknown. The purpose of this study was to evaluate the effect of IR on differential vascular remodeling after balloon angioplasty in porcine coronary arteries. Methods: A total of 20 juvenile swine (30 coronary arteries) were subjected to overstretch balloon injury (BI) followed by IR with either β- or γ-radiation ( 90 Y or 192 Ir). After 2 weeks following treatment, serial tissue sections were perfusion fixed and stained by hematoxylin and eosin (HE control: 3.6±0.7 mm 2 ). In contrast, the DReg was increased following IR, as demonstrated by the FL and the calculated area of the injured segment (control: 2.7±0.5 mm 2 ; β: 5.5±1.1 mm 2 , γ: 5.5±1.1 mm 2 , P Conclusion: We consider that the global arterial remodeling after IR is a heterogeneous process that includes the absence of retraction in an UReg and a positive remodeling in the DReg as shown in the porcine coronary model. These changes in adventitia and neointima appear to contribute to differential vascular remodeling caused by IR in injured vessels.

Yttrium-90 delivered via a centering catheter and afterloader, given both before and after stent implantation, inhibits neointima formation in porcine coronary arteries

BACKGROUND Intracoronary radiation (IR) studies have shown reduction of neointima formation (NF). Extrapolation of animal studies with beta-radiation to clinical trials have shown variable results, which may be related to dosimetry, centering issues, and/or shielding of beta-rays by the stent metal. We examined the effect of yttrium-90 (90Y), a pure beta-emitter delivered via an automatic afterloader to a centering catheter, on the inhibition of NF in balloon-injured (BI) porcine coronary arteries as well as in arteries receiving 90Y either prior to or following stent implantation (SI). METHODS Twenty-three swine (44 coronary arteries) were studied. In the first study, IR (18 Gy at 1.2 mm from the balloon surface) was administered in 17 arteries following BI, while eight control arteries were subjected to BI only. In the second study, 10 swine (19 coronary arteries) underwent SI. IR (18 Gy) was administered in six arteries before and in eight arteries after SI, while five control arteries received SI only. The animals were sacrificed 2 weeks after BI and 4 weeks after SI. Their coronaries were perfusion fixed and stained, and vessel parameters (intimal area [IA] and medial fracture length [FL]) were analyzed by computer-aided histomorphometry. RESULTS Arteries subjected to IR following BI had less NF compared to controls (IA/FL = 0.14 +/- 0.2 mm vs. 0.49 +/- 0.2 mm; P = 0.003). IA was reduced significantly in the arteries receiving radiation before and after SI compared to controls (0.92 +/- 0.98 and 0.00 +/- 0/00 vs. 2.72 +/- 1.2 mm2; P = 0.014), despite similar SI in all groups. CONCLUSIONS IR with 90Y delivered via a centering catheter is safe and effective with complete and homogenous inhibition of NF in the context of BI or SI in the porcine coronary model.

Anti-restenotic effect of copper-62 liquid-filled balloon in porcine coronary arteries: novel use of a short half-life positron emitter

PURPOSE To determine the efficacy of the use of copper-62, a positron emitter with a half-life of 9.7 minutes, as an intracoronary brachytherapy (IRBT) source in the prevention of neointima formation (NF) following overstretch balloon injury (BI) in the porcine model. METHODS AND MATERIALS Sixteen swine were treated after BI to their left anterior descending (LAD), left circumflex (LCX), and/or right coronary artery (RCA). Twelve of the injured arteries received placebo and 10 received 25 Gy, delivered to 0.5 mm from the surface of the treatment balloon filled with liquid (62)Cu. Dosimetry was based on Monte Carlo calculations. Two weeks after treatment, the animals were sacrificed, and the treated coronaries were perfusion-fixed and stained. Intimal area (IA) and medial fracture length (FL) were analyzed by computer-aided histomorphometry. RESULTS The ((62)Zn/(62)Cu) generator, together with a rapid concentration process, was successful in delivering the short-lived (62)Cu at the high concentration required for intravascular brachytherapy (IVBT). The fracture length in the two groups was similar (2.10 +/- 0.57; 2.02 +/- 0.77; p = NS). Arteries studied showed significant reduction in NF (IA: 0.23 +/- 0.47 mm(2) vs. 1.08 +/- 0.57 mm(2); p < 0.01. IA/FL = 0.09 +/- 0.17 mm vs. 0.51 +/- 0.21 mm; p < 0.01). CONCLUSIONS This study demonstrated that use of liquid (62)Cu as an IVBT source is safe and feasible. All 16 swine tolerated the treatment well with no radiation-induced side effects or symptoms throughout the 2-week period. The isotope delivered the dose necessary to inhibit NF in the porcine coronary BI model.

Vascular repair after balloon overstretch injury in porcine model effects of intracoronary radiation.

OBJECTIVES The purpose of this study was to evaluate the effect of IR on thrombus formation and dissection repair following overstretch balloon injury in porcine coronary arteries. BACKGROUND Exposure of blood to the injured arterial wall after percutaneous transluminal coronary angioplasty (PTCA) induces thrombus formation and inflammation in the dissection plane. Neointima formation is related to smooth muscle cell (SMC) proliferation and migration into the preformed thrombus. Intracoronary radiation (IR) with doses of 10 to 25 Gy using either beta or gamma emitters can prevent neointima accumulation by reducing SMC proliferation. However, there are some indications that IR may delay the process of dissection repair after PTCA. The purpose of this study was to evaluate the effect of IR on thrombus formation and dissection repair after overstretch balloon injury in porcine coronary arteries. METHODS Forty porcine coronaries were injured by balloon overstretch followed by either 0 or 18 Gy of 90Y prescribed to 1.2 mm from the balloon center. The animals were euthanized 14 days after treatment, and intimal area (IA) and IA corrected for medial fracture length (IA/FL) were quantified by digital image analysis. Dissections were quantified by tracing the length, thickness and area behind the dissection flap. The rate of dissections was calculated for each group. Thrombi were identified and designated as intraluminal thrombus or thrombus within dissection planes (mural thrombus), and area measurements were obtained. RESULTS The irradiated group showed a significant reduction of IA/FL (0.55 +/- 0.29 vs. 0.05 +/- 0.09; p < 0.001). No difference was observed in the rate of dissection between control and irradiated arteries (77% vs. 88%, respectively). The control group showed a smaller dissection area (0.19 +/- 0.28 mm2 vs. 0.32 +/- 0.29 mm2; p < 0.05) with smaller mural thrombi (0.03 +/-0.01 mm2 vs. 0.29 +/- 0.30 mm2; p < 0.001). A strong correlation between dissection area and neointima area was observed only in the control group (R2 = 0.474; p < 0.003; alpha0.05 = 0.862). A positive correlation between mural thrombus and dissection area was observed only in the irradiated group (R2 = 0.889; p < 0.001; alpha0.05 = 1.00). CONCLUSIONS These results suggest that the dissection area may be a useful parameter by which to quantify the extent of injury and repair after IR and may indicate an incomplete healing process after IR at this time point.