Elective removal of an intramyocardial bullet.

A 26-year-old man had a gunshot wound in the right posterolateral aspect of the chest. A chest radiograph showed the bullet in the region of the cardiac silhouette. The patient was hemodynamically stable and had no complaints of dyspnea or abdominal pain. Echocardiography and computed tomography identified the bullet in the wall of the right ventricle. The surgical management of the injury is discussed in detail.

Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and Cdc2 kinase inhibit graft coronary artery disease.

BACKGROUND: The long-term success of cardiac transplantation is limited by graft coronary artery disease (GCAD). Antisense oligonucleotides (ASs) to proliferating cell nuclear antigen (PCNA) and Cdc2 kinase (Cdc2 k) can arrest cell cycle progression and inhibit neointimal hyperplasia. Transforming growth factor-ss(1) (TGF-ss(1)) has been implicated in vascular smooth muscle cell (VSMC) activation. The role of TGF-ss(1) in GCAD remains unclear. We hypothesized that ASs to PCNA and Cdc2 k would inhibit VSMC proliferation and GCAD. METHODS AND RESULTS: In vitro VSMC proliferation was determined after pretreatment with AS solution or medium alone followed by angiotensin II stimulation. PVG-to-ACI rat heterotopic cardiac transplantation procedures were performed after ex vivo pressure-mediated transfection of ASs to PCNA and Cdc2k or saline alone. At postoperative days 30, 60, and 90, allografts were assessed for GCAD, percent neointimal macrophages and VSMCs, and TGF-ss(1) activity. AS pretreatment significantly attenuated VSMC proliferation. At postoperative day 90, percent affected arteries, percent occlusion, and intima-media ratio demonstrated severe GCAD in saline-treated allografts, whereas these parameters were significantly lower in AS-treated allografts. Percent neointimal macrophages and VSMCs was reduced in AS-treated allografts. TGF-ss(1) activity was increased in saline compared with AS-treated allografts and nontransplanted heart controls. CONCLUSIONS: ASs to PCNA and Cdc2 k inhibit VSMC proliferation in vitro and reduce GCAD, percent neointimal VSMCs and macrophages, and TGF-ss(1) activity in vivo. TGF-ss(1) may play a "response to injury" role in the development of GCAD. The prevention of GCAD via AS inhibition of cell cycle regulatory genes before reperfusion may offer a useful clinical alternative to current therapeutic strategies.

Effects of humanized monoclonal antibody to rhesus CD11a in rhesus monkey cardiac allograft recipients.

INTRODUCTION: Leukocyte function-associated antigen-1 (LFA-1, CD11a) monoclonal antibody (mAb) affects many leukocyte functions without cell depletion. We hypothesized that the use of a humanized, anti-rhesus modified LFA-1 mAb (H2C12) in rhesus monkeys would cause: (1) prolonged heart allograft survival, (2) inhibition of primary but not secondary antibody responses, and (3) minimal drug toxicity. METHODS AND RESULTS: Control (n=5) and H2C12-treated (n=7) (8-20 mg/kg i.v. on day -1 followed by 10 mg/kg/day) adult male rhesus recipients were inoculated with GP120 protein antigen on day -28 and -1 and grafted with heterotopic abdominal hearts (day 0). Donor-recipient pairs were equally MLR mismatched (4329.8+/-1124.1 CPM controls vs. 7289.0+/-1926.5 treated, P=NS). Mean heart allograft survival as evaluated by daily abdominal palpation was significantly prolonged in high dose recipients (23.0+/-2.6, n=4) vesus controls (8.2+/-1.3, n=5, P<0.02, Mann-Whitney U test). H2C12 treatment did not produce signs of cytokine release or toxicity, was nondepleting, but down-modulated PBL CD11a expression to 43.4+/-3.6% (n=4) of control levels (n=5) at day 7 as demonstrated by flow cytometry. It had no effect on postoperative Con A or MLR and did not prevent mAb clearance due to the rhesus-antihuman antibody response. The addition of mycophenolate mofitil prevented rhesus-antihuman antibody response with therapeutic H2C12 levels seen for >35 days. CONCLUSIONS: The use of this mAb to block CD11a had the benefit of being a well tolerated, highly targeted therapy. These are the first results showing that monotherapy with anti-leukocyte function-associated antigen-1 mAb prolonged survival of MLR mismatched allogenic cardiac grafts in primates.

Optimization of ex vivo pressure mediated delivery of antisense oligodeoxynucleotides to ICAM-1 reduces reperfusion injury in rat cardiac allografts.

BACKGROUND: Our purpose was to optimize hyperbaric pressure as a vector for ex vivo transfection of antisense oligodeoxynucleotides (AS-ODN) to intercellular adhesion molecule-1 to limit reperfusion injury (RI) in cardiac allografts. We investigated the effects of increased pressure, incubation time, and AS-ODN concentrations on transfection efficiency and toxicity. METHODS AND RESULTS: PVG (RT1c) donor hearts were heterotopically transplanted to ACI (RT1a) recipients. Donor hearts were harvested and the various groups were treated at: (1) different pressure (1-9 atm) for 45 min with 80 micromol/liter AS-ODN; (2) different incubation times (15 min to 6 hr) at 5 atm with 80 micromol/liter AS-ODN; 3) different AS-ODN concentrations (80-240 micromol/liter) at 5 atm for 45 min. Hearts were procured 24 or 72 hr after transplantation. Transfection efficiency was determined with fluorescein-labeled AS-ODN. The degree of RI was determined with biochemical and histological analysis. Increasing pressure from ambient (1 atm) pressure to pressures as high as 9 atm leads to a increase in transfection efficiency from 1.7+/-.5 to 62+/-3.9% and a reduction in RI. Increased incubation time up to 45 min increased transfection efficiency and reduced RI, but longer incubation times induced significant toxicity to the allograft. Increased AS-ODN concentrations improved transfection and reduced RI. CONCLUSIONS: Hyperbaric pressure is a safe and effective vector for the ex vivo delivery of AS-ICAM-1-ODN to rodent cardiac allografts and results in a reduction in reperfusion injury.

ICAM-1 affects reperfusion injury and graft function after cardiac transplantation.

BACKGROUND: The effects of increased expression of intercellular adhesion molecule (ICAM-1), an important mediator of neutrophil-mediated reperfusion injury (RI), were assessed in donor cardiac allografts in a heterotopic rat transplantation model. METHODS: At -24 h, PVG donors were untreated (n = 35) or treated (n = 37) with lipopolysaccharide (LPS, 5 mg/kg ip). Hearts were procured at 0 h, stored at 4 degrees C for 45 min, and grafted heterotopically into ACI recipients pretreated with vehicle or anti-ICAM-1 (1A29) mAb. Intracardiac balloons (n = 8 per group) were used to measure allograft left ventricular function (dP/dt) prior to harvest and following reperfusion. RI was assessed at 6, 12, and 24 h by myeloperoxidase (MPO) levels, percentage wet weight (%w/w), and percentage contraction band necrosis (%CBN). RESULTS: At 12 h, LPS-pretreated grafts showed increased ICAM-1 expression by Northern blot (n = 3) and immunohistochemistry (n = 3) and significantly increased MPO (0.33 +/- 0.2 U/mg vs 0.05 +/- 0.04 U/mg at 12 h), %w/w (81.7 +/- 1.8% vs 79.2 +/- 0.7% at 12 h), and %CBN (15.2 +/- 1. 9% vs 11.4 +/- 2.0% at 24 h). LPS pretreatment had no effect on graft function at early time points (baseline to 2 h) but led to depressed dP/dt at later time points with trends toward significance at 12 h (2101 +/- 1653 mmHg/s vs 173 +/- 201 mmHg/s, P = 0.06, ANOVA). Recipient 1A29 treatment (n = 6 per group) reversed the effects of LPS pretreatment in all three RI parameters and significantly improved functional recovery. CONCLUSIONS: Alteration of cardiac graft phenotype to that likely seen in clinical organ donors leads to increased delayed-onset myocardial RI following transplantation in this model. The blockade of this increased RI following 1A29 mAb treatment supports a central role for ICAM-1 in this process.

Antisense oligodeoxynucleotides prevent acute cardiac allograft rejection via a novel, nontoxic, highly efficient transfection method.

BACKGROUND: We hypothesized that ex vivo donor allograft transfection with antisense oligodeoxynucleotide (AS ODN) would inhibit the expression of intercellular adhesion molecule (ICAM)-1, an important mediator of T-cell adhesion and costimulation, and therefore suppress acute cardiac rejection. METHODS: Hearts were transfected ex vivo with AS, reverse AS ODN, or saline by applying 3 atm pressure for 45 min at 4 degrees C. Grafts were then transplanted into allogenic recipients +/- treatment with leukocyte function-associated antigen (LFA)-1 monoclonal antibody (mAb) (1.5 mg/kg intravenously), cyclosporine (2.5 mg/ kg/day p.o.), or rapamycin (0.025 mg/kg/day intraperitoneally). Reperfusion injury was assessed in grafts harvested at early time points using the myeloperoxidase, %wet weight, and %contraction band necrosis assays; transfection efficiency was assessed using fluorescent microscopy; and efficacy of ICAM-1 blockade was assessed using immunohistochemistry. Other grafts were followed until rejection with donor/third-party skin grafting, adoptive transfer, and interleukin 2 infusion studies in selected recipients. RESULTS: Transfection was highly efficient (fluorescein isothiocyanate-ODN in 48+/-5% of total myocardial nuclei), nontoxic, and reduced the ICAM-1-positive area to 53+/-14% versus having no effect on MHC class I expression (n=4). The incidence of survival >60 days after AS ODN + LFA-1 monoclonal antibody was 75%, significantly higher than other regimens. CONCLUSION: AS ODN hyperbaric transfection proved highly efficient, effective at ICAM-1 blockade, and induced cardiac allograft tolerance when combined with LFA-1 monoclonal antibody. This highly targeted alteration of allograft immunogenicity may have an important role in future immunosuppressive strategies.

Pressure delivery of AS-ICAM-1 ODN with LFA-1 mAb reduces reperfusion injury in cardiac allografts.

BACKGROUND: The goal of this study is to determine the effects of ex vivo hyperbaric pressure administration of AS-ICAM-1 ODN and systemic anti-LFA-1 mAb treatment on reperfusion injury in the rat cardiac allograft model. METHODS: A PVG to ACI functional heterotopic rat heart model was used. Donor hearts were treated with either saline or AS-ICAM-1 ODN and 5 atm of hyperbaric pressure for 45 minutes. Anti-LFA-1 mAb was administered systemically prior to reperfusion of the allograft. Allografts were procured 24 hours after transplantation for assessment of reperfusion injury or 72 hours to determine ICAM-1 protein expression. RESULTS: Ex vivo administration of AS-ICAM-1 ODN led to decreases in percentage wet weight (77.1+/-0.83% vs 78.7+/-1.0%, p < 0.05), myeloperoxidase activity (3.14+/-0.72 vs 4.07+/-0.59, p < 0.05), contraction band necrosis (6.4+/-6.47% vs 21.1+/-7.43%, p < 0.01), and ICAM-1 protein expression determined by immunohistochemistry compared to saline controls. Treatment with anti-LFA-1 mAb resulted in decreases in wet weight ratio (76.7+/-0.63%, p < 0.05 vs saline), myeloperoxidase activity (3.58+/-0.39, p < 0.05 vs saline) and contraction band necrosis (11.8+/-3.56%, p < 0.05 vs saline). Combination of pressure administration of AS-ICAM-1 ODN and anti-LFA-1 mAb decreased wet weight ratios (77.1+/-0.93%, p < 0.05 vs saline), myeloperoxidase activity (2.88+/-0.44, p < 0.01 vs saline), and contraction band necrosis (6.75+/-5.67%, p < 0.05 vs saline). CONCLUSIONS: Ex vivo pressure mediated delivery of AS-ICAM-1 ODN decreases ICAM-1 protein expression, reduces reperfusion injury in rodent cardiac allografts, and is more effective than anti-LFA-1 mAb treatment alone.

Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model.

BACKGROUND: Chronic graft vascular disease (CGVD) in cardiac allografts has been defined as a slowly evolving vasculopathy unresponsive to conventional immunosuppression. We compared 4 rodent models of CGVD to evaluate the reproducibility of CGVD in heart allografts. Rapamycin (Rapa) and cyclosporine (CSA) were then used to treat CGVD. METHODS AND RESULTS: Hearts were harvested and placed heterotopically into allogenic recipients. CGVD scores of PVG allografts from ACI recipients treated with CSA on days 1 through 10 were significantly elevated on day 90 (n=16) compared with other models (immunosuppression used): (1) Lewis to F344 recipients (CSA), (2) Brown Norway to Lewis (FK506), and (3) DA to Wistar-Firth (methylprednisolone, azathioprine, CSA). Although delayed (day 60 to 90) CSA treatment had no effect (n=6), delayed Rapa (3 mg. kg-1. d-1 IP) reversed CGVD in PVG grafts (0.22+/-0.19 on day 90, n=6). ACI isografts showed no evidence of CGVD (n=6) at day 90. Immunohistochemistry of PVG grafts revealed perivascular infiltrates consisting of CD4(+) T cells and limited numbers of macrophages persisting up to day 90. Flow cytometry demonstrated increased levels of anti-donor antibody at day 90, which was significantly inhibited by Rapa treatment. CONCLUSIONS: PVG grafts developed a significant increase in CGVD without evidence of ongoing myocardial rejection. This CGVD appeared to be mediated by both cellular and humoral mechanisms, given CD4(+) perivascular infiltrates and increased levels of anti-donor antibody. The anti-CGVD effectiveness of Rapa during a period in which there was little myocardial cellular infiltrate supports a novel mechanism of effect such as smooth muscle or B-cell inhibition.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues.

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Ex vivo gene therapy prevents chronic graft vascular disease in cardiac allografts.

OBJECTIVE: We hypothesized that ex vivo hyperbaric transfection of antisense oligodeoxynucleotides for blockade of intercellular adhesion molecule-1, an important mediator of cell adhesion and T-cell co-stimulation, would reduce chronic graft vascular disease in cardiac allografts. METHODS: PVG hearts underwent ex vivo transfection with antisense, reverse antisense intercellular adhesion molecule-1 oligodeoxynucleotide (80 micromol/L), or saline solution at 3 atm pressure for 45 minutes at 4 degrees C and were transplanted heterotopically into ACI recipients with or without treatment with intercellular adhesion molecule-1 (1A29) or leukocyte function associated antigen-1 (WT.1) monoclonal antibodies. Transfection efficiency was confirmed with fluorescein isothiocyanate-labeled oligodeoxynucleotides and fluorescent microscopy. Efficacy of intracellular adhesion molecule-1 blockade was assessed with the use of immunohistochemistry. Graft reperfusion injury was evaluated at 6 to 24 hours by neutrophil infiltration (myeloperoxidase [MPO]), cardiac edema (%wt/wt), and histologic injury (percent contraction band necrosis). Grafts from recipients treated with cyclosporine A (5 mg/kg per day, days 0 to 9) were scored for chronic graft vascular disease on postoperative day 90 ranging from 0 (no involvement) to 4 (>50% vascular occlusion). RESULTS: Transfection was highly efficient (fluorescein isothiocyanate-labeled oligodeoxynucleotides in 48%+/-5% of total myocardial nuclei) and effective at blocking intracellular adhesion molecule-1 expression (positive area in allografts taken on postoperative day 3 was reduced from 100%+/-0% to 52%+/-14%, n=4). Blockade with antisense oligodeoxynucleotides versus monoclonal antibodies was less effective at preventing reperfusion injury while more effective at reducing chronic graft vascular disease (score 0.98+/-0.48, p < 0.05). Reverse antisense oligodeoxynucleotides and vector control (antisense oligodeoxynucleotide infusion without pressure) groups failed to demonstrate this beneficial effect. CONCLUSION: Hyperbaric transfection of antisense oligodeoxynucleotides proved highly efficient, effective at blockade of intracellular adhesion molecule-1, and demonstrated a sequence-specific reduction in chronic graft vascular disease. This highly targeted alteration of donor organ immunogenicity may have an important future role in clinical immunosuppressive strategies.

Prevention of ischemically induced neointimal hyperplasia using ex vivo antisense oligodeoxynucleotides.

BACKGROUND: Chronic graft vascular disease in cardiac allografts results from coronary artery neointimal formation. Vascular ischemia has been shown to provoke the development of neointimal hyperplasia through endothelial damage. We used a rodent model of neointimal formation to test the in vivo effects of antisense oligodeoxynucleotides (AS ODN) specifically designed to block this process. METHODS: Aortas from ACI rats were mock transfected or transfected with 18 base pair AS ODNs against the 5' start codon region of both CDC2 kinase, and proliferating cell nuclear antigen (PCNA) mRNA. Transfection was accomplished by placing the aorta in ODN solution (transfected group, 40 micromol/L of each sequence) or saline solution alone (mock-transfected group) and exposing to hydrostatic pressure (2 atm) for 24 hours at 4 degrees C. Vessels were then interposition-grafted into the abdominal aorta of untreated isogenic recipients and procured on postoperative days (POD) 1, 6, and 14. RESULTS: Nuclear localization of fluorescein isothiocyanate ODN was observed in 81%+/-3% of medial smooth muscle cells at 24 hours after interposition grafting and reperfusion. Efficacy of AS ODNs at blocking CDC2 kinase and PCNA was verified on POD 6 by enzyme-linked immunosorbent assay. This blockade significantly reduced ischemically induced vascular narrowing on POD 14 as assessed by use of computerized image analysis (3.85%+/-2.45% luminal narrowing for transfected vs 7.11%+/-2.03% for control subjects, p < 0.03). CONCLUSIONS: Our data show the efficacy of AS ODN at blocking rat PCNA and CDC2 kinase up-regulation provoked by ischemia. This ex vivo approach had beneficial effects against vascular narrowing in a rodent model of ischemically induced neointimal hyperplasia, an antigen-independent factor important in the development of subsequent chronic graft vascular disease.

Effects of increased ICAM-1 on reperfusion injury and chronic graft vascular disease.

BACKGROUND: The purpose of this study was to assess the impact of increased donor cardiac intercellular adhesion molecule (ICAM-1) expression on both reperfusion injury and chronic graft vascular disease after transplantation. METHODS: Hearts were harvested from donor rats before and after pretreatment with lipopolysaccharide at -24 hours, underwent 45 minutes of cold ischemia, and were transplanted into ACI recipients with or without anti-ICAM-1 monoclonal antibody treatment. Grafts were procured early for analysis of ICAM-1 expression and reperfusion injury or the recipients were treated with cyclosporin A (to allow long-term graft acceptance) for postoperative days 0 through 9 with procurement on postoperative day 90 to histologically score for chronic graft vascular disease. RESULTS: Lipopolysaccharide-pretreated PVG heart grafts showed increased ICAM-1 expression by Northern blot and immunohistochemical analysis leading to increased reperfusion injury as assessed by neutrophil infiltration (myeloperoxidase), cardiac edema (percentage wet weight), and histologic injury (percentage area of contraction band necrosis), which was reversed by recipient treatment with anti-ICAM-1 monoclonal antibody. After administration of cyclosporin A, 5 mg/kg for 10 days, lipopolysaccharide-treated grafts had significantly worse chronic graft vascular disease scores (2.56 +/- 0.57 versus 1.84 +/- 0.75; p < 0.05 by Mann-Whitney U test). CONCLUSIONS: The induction donor inflammatory state before harvest leading to increased cardiac ICAM-1 expression promotes reperfusion injury and chronic graft vascular disease after transplantation in this rodent heterotopic heart model.

Platelet activating factor inhibition reduces lung injury after cardiopulmonary bypass.

Because cardiopulmonary bypass (CPB) produces a diffuse inflammatory reaction that may injure multiple organs and complicate cardiac surgical procedures, we examined the use of a competitive inhibitor of platelet activating factor (SDZ HUL-412) in a porcine model of CPB as a means to ameliorate pulmonary injury after CPB. Thirteen pigs (35 to 40 kg) underwent CPB at 28 degrees C for 2 hours, followed by 2 hours of observation. Group I (n = 6) received SDZ HUL-412 (a quinolinium compound) intravenously (3 mg/kg loading dose and 2 mg.kg-1.h-1 continuous infusion) starting before sternotomy. Group II (n = 7) received a saline vehicle. Peak airway pressure, pulmonary arterial pressure, left atrial pressure, and arterial blood gases were measured and flow cytometry evaluated surface expression of adhesion molecule subunit CD18 on circulating neutrophils. Pulmonary function was significantly improved in group I. Fifteen minutes after CPB, dynamic lung compliance in group I was 91% +/- 12% of baseline versus 49% +/- 5.2% in group II (p = 0.06 by analysis of variance). After CPB, the arterial oxygen pressure was also significantly better in group I than in group II (425 +/- 61 versus 234 +/- 76 mm Hg) (p < 0.05). The rise in pulmonary vascular resistance after CPB was less in group I (p < 0.05) (323 +/- 55 to 553 +/- 106 dynes.s.cm-5) than in group II (531 +/- 177 to 884 +/- 419 dynes.s.cm-5) at the end of the observation period. CD18 up-regulation increased similarly in the two groups during CPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Two decades of coronary artery bypass graft surgery in young adults.

Between January 1970 and December 1991, 201 patients < or = 40 years of age underwent coronary artery bypass graft surgery (CABG). Group 1 (1970 to 1980, n = 119) and group 2 (1981 to 1991, n = 82) corresponded to the eras before and after the onset of percutaneous transluminal coronary angioplasty (PTCA), respectively, and were analyzed for trends in patient profile, treatment, and risk factors for coronary artery disease (CAD): smoking, hypertension, hypercholesterolemia, diabetes, and family history. Mean age at operation was similar in the groups (1, 37 +/- 3.4 years; 2, 36 +/- 3.1 years). Women made up 18% of group 1 and 27% of group 2 (P = .048). Risk factor profile differed in the two groups: group 1 had more smokers (80%) than group 2 (68%) (P = .085), fewer patients with hypercholesterolemia (1, 37%; 2, 52%; P = .065), and significantly fewer diabetics (1, 10%; 2, 25%; P < .043). Mean preoperative New York Heart Association (NYHA) class was 3.2 in group 1 and 3.0 in group 2. The distributions of single-, double-, and triple-vessel CAD were similar in the groups. Preoperative myocardial infarction occurred in 55% of group 1 versus 61% in group 2 (P = NS). No group 1 patient received PTCA before CABG, but PTCA was performed in 15 group 2 patients. Left internal mammary artery grafts were used in 4% of group 1 and 57% of group 2 patients. CABG operative mortality was 7.0% in group 1 and 1.2% in group 2.(ABSTRACT TRUNCATED AT 250 WORDS)