Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy.

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

Angiogenic effects despite limited cell survival of bone marrow-derived mesenchymal stem cells under ischemia.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

Trends in invasive disease due to Candida species following heart and lung transplantation.

Although invasive candidiasis (IC) causes significant morbidity and mortality in patients who undergo heart, lung, or heart-lung transplantation, a systematic study in a large cohort of thoracic organ transplant recipients has not been reported to date. Clinical and microbiological data were reviewed for 1305 patients who underwent thoracic organ transplantation at Stanford University Medical Center between 1980 and 2004. We identified and analyzed 76 episodes of IC in 68 patients (overall incidence 5.2% per patient).The incidence of IC was higher in lung (LTx) and heart-lung transplant (HLTx) recipients as compared with heart transplant (HTx) recipients (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.1-2.7).The incidence of IC decreased over time in all thoracic organ transplant recipients, decreasing from 6.1% in the 1980-1986 time period to 2.1% in the 2001-2004 era in the HTx recipients, and from 20% in the 1980-1986 period to 1.8% in the 2001-2004 period in the LTx and HLTx recipients.The most common site of infection differed between the HTx and LTx cohorts, with bloodstream or disseminated disease in the former and tracheobronchitis in the latter. IC in the first year after transplant was significantly associated with death in both HTx (RR 2.9, 95% CI 1.8-4.6, P=0.001) and LTx and HLTx patients (RR 3.0, 95% CI 1.9-4.6, P<0.001). The attributable mortality from IC decreased during the 25-year period of observation, from 36% to 20% in the HTx recipients and from 39% to 15% in the LTx and HLTx recipients. There were a significant number of cases caused by non-albicans Candida species in all patients, with a trend toward higher mortality in the HTx group. In conclusion, the incidence and attributable mortality of IC in thoracic organ transplant recipients has significantly declined over the past 25 years.The use of newer antifungal agents for prophylaxis and treatment, the decrease in the incidence of cytomegalovirus disease, and the use of more selective immunosuppression, among other factors, may have been responsible for this change.

Twenty-year survivors of heart transplantation at Stanford University.

Human heart transplantation started 40 years ago. Medical records of all cardiac transplants performed at Stanford were reviewed. A total of 1446 heart transplantations have been performed between January 1968 and December 2007 with an increase of 1-year survival from 43.1% to 90.2%. Sixty patients who were transplanted between 1968 and 1987 were identified who survived at least 20 years. Twenty-year survivors had a mean age at transplant of 29.4 +/- 13.6 years. Rejection-free and infection-free 1-year survivals were 14.3% and 18.8%, respectively. At their last follow-up, 86.7% of long-term survivors were treated for hypertension, 28.3% showed chronic renal dysfunction, 6.7% required hemodialysis, 10% were status postkidney transplantation, 13.3% were treated for diabetes mellitus, 36.7% had a history of malignancy and 43.3% had evidence of allograft vasculopathy. The half-life conditional on survival to 20 years was 28.1 years. Eleven patients received a second heart transplant after 11.9 +/- 8.0 years. The most common causes of death were allograft vasculopathy (56.3%) and nonlymphoid malignancy (25.0%). Twenty-year survival was achieved in 12.5% of patients transplanted before 1988. Although still associated with considerable morbidity, long-term survival is expected to occur at much higher rates in the future due to major advances in the field over the past decade.

Is the malononitrilamide FK778 better for the prevention of acute or chronic rejection?

OBJECTIVE: The aim of this study was to assess the efficacy of FK778 to prevent acute and chronic allograft rejection compared with other immunosuppressive agents. MATERIALS AND METHODS: Heterotopic Brown-Norway (BN)-to-Lewis rat cardiac transplantations and heterotopic BN-to-Lewis tracheal transplantations were performed to study acute heart rejection and the development of chronic obliterative airway disease (OAD), respectively. Recipients were treated with FK778, tacrolimus, MMF, or sirolimus for 10 days (acute rejection study) or 28 days (chronic OAD study) at varying doses. RESULTS: In untreated recipients, cardiac allograft survival was 6.2 +/- 0.4 days. FK778 (20 mg/kg), tacrolimus (2 or 8 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly prolonged graft survival to 17.0 +/- 2.8, 18.5 +/- 2.7, 25.0 +/- 2.5, 20.7 +/- 3.8, 14.5 +/- 2.2, and 23.2 +/- 1.5 days, respectively (P < .05). Tracheal grafts in untreated recipients showed intense infiltration and complete luminal obliteration by day 28. FK778 (20 mg/kg), tacrolimus (1 or 4 mg/kg), MMF (10 or 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly inhibited tracheal luminal obliteration (19.5% +/- 16.4%, 44.2% +/- 33.6%, 12.3% +/- 3.3%, 61.7% +/- 18.6%, 18.3% +/- 11.3%, 55.0% +/- 30.9%, and 8.5% +/- 3.5% (P < .05). All 4 high-dose groups showed similar efficacy. CONCLUSIONS: When used in therapeutic doses, tacrolimus and sirolimus were more effective than FK778 to prolong cardiac allograft survival. However, with its antiproliferative effects on smooth muscle cells, its good tolerability, and its blockade of cytomegalovirus replication, FK778 proved effective to prevent chronic OAD development. Thus, FK778 may acquire an important role in maintenance therapy for the prevention of long-term fibroproliferative complications.

Stem cell transplantation: the lung barrier.

BACKGROUND: Mesenchymal stem cells (MSCs) show differentiation capacity along mesenchymal lineages and have the potential to aid tissue regeneration. MSC transplantation strategies are therefore currently being assessed following injury to various organs. However, potential MSC migration to these organs after intravenous (IV) MSC injection continues to be impeded by cell trapping within the lung. METHODS: Mouse MSCs were isolated, purified, transfected with firefly luciferase, and labeled with CSFE. Their size was assessed in vitro. To estimate the diameter of mouse pulmonary capillaries, fluorescence-labeled microspheres of different sizes were injected with or without sodium nitroprusside (SN) pretreatment. The lungs were harvested after 30 seconds and mean numbers of trapped microspheres per high-power field (HPF) were calculated. After IV injection of MSC suspensions (with or without SN), their dynamic distribution was monitored by in vivo luciferine imaging as well as by histopathology. RESULTS: The diameter of suspended MSCs in vitro was 15 to 19 microm. Whereas nearly no 4-microm microspheres could be detected in lung sections, the numbers of trapped 10- and 15-microm microspheres could be significantly decreased by prior SN injection from 19.3 +/- 11.1 to 6.0 +/- 1.6 cells/HPF (P = .004) and from 34.9 +/- 11.9 to 25.6 +/- 8.1 cells/HPF (P = .028), respectively. Within seconds after MSC IV injection, the vast majority of cells was found in the lungs. However, cell trapping in the pulmonary microvasculature was significantly reduced by pre-treatment with SN. CONCLUSIONS: We demonstrate that cell trapping in lungs can be reduced with IV SN pretreatment, increasing MSC passage through the lung capillaries, and potentially facilitating cell access to injured organs.

Reduction of vein graft disease using photodynamic therapy with motexafin lutetium in a rodent isograft model.

BACKGROUND: Motexafin lutetium (Lu-Tex) is a photosensitizer that targets atheromatous plaque. Subsequent photoactivation (photodynamic therapy [PDT]) induces local cytotoxic effects. The aim of the present study was to investigate whether Lu-Tex targets vein graft intimal hyperplasia and whether subsequent photoactivation attenuates the disease process. METHODS AND RESULTS: The subcellular localization of Lu-Tex and postillumination viability were studied in cultured human vein graft smooth muscle cells. Inferior vena cava-grafted rats were injected with Lu-Tex (10 mg/kg) 4 or 12 weeks after grafting. Biodistribution was assessed in a subgroup 24 hours after administration. Light therapy (742 nm) was performed 24 hours after Lu-Tex injection by illuminating intraperitoneally placed isografts using a laparotomy. Animals were divided into the following 4 groups: PDT (n=15), Lu-Tex injection and laparotomy (n=13), light treatment (n=14), and laparotomy only (n=13). Grafts were harvested 14 days after treatment for histochemical analysis. Lu-Tex localized within subcellular organelles of smooth muscle cells, and subsequent photoactivation induced cell death via apoptosis. The Lu-Tex concentrations present in the vein grafts were 9.3 times higher than those in the normal inferior vena cava. Postoperative PDT at 4 weeks after surgery significantly reduced the intima/media ratio, whereas treatment at 12 weeks did not reduce the intima/media ratio. Activated macrophages were observed 4 weeks after grafting; however, a significant reduction occurred in these cells by 12 weeks. The mechanism by which PDT works may be related to the presence of activated macrophages. CONCLUSIONS: PDT significantly reduces the intima/media ratio in the early phase of vein graft disease. Lu-Tex-mediated PDT may be a viable method for the attenuation of atherosclerotic disease in vein grafts.

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.

Upregulation of Bcl-2 through caspase-3 inhibition ameliorates ischemia/reperfusion injury in rat cardiac allografts.

BACKGROUND: Oxidative stress after ischemia/reperfusion of cardiac allografts leads to cytokine production. Bcl-2, an inhibitor of apoptosis, also has strong antioxidant properties. Caspase-3 is known to cleave bcl-2. This study tests the hypothesis that bcl-2 is downregulated while tumor necrosis factor-alpha (TNF-alpha) levels increase after cardiac transplantation. Furthermore, the use of caspase-3 inhibition was investigated as a strategy for preserving myocardial bcl-2 and mitochondrial cytochrome c after transplantation. METHODS AND RESULTS: PVG-to-ACI rat heterotopic cardiac transplantations were performed in 4 groups designed with 30 minutes' ischemia and 4 or 8 hours of reperfusion (n=4 per group). Treatment consisted of DEVD-CHO 500 microgram IP per animal to donor and recipient 2 hours before transplantation and 250 microgram IC into allograft. Controls were treated with saline. Grafts were analyzed by reverse transcription-polymerase chain reaction for bcl-2 mRNA, by ELISA for TNF-alpha, for myeloperoxidase activity, and by Western blot for cytochrome c. In untreated groups, bcl-2 mRNA decreased significantly over time, whereas TNF-alpha increased significantly at 4 hours (P=0.003) and returned to baseline after 8 hours' reperfusion (P=NS compared with normal hearts). Treatment with caspase-3 inhibitor showed significant upregulation of bcl-2 mRNA expression after 4 and 8 hours of reperfusion (P<0.001 versus control), with a concomitant decrease in TNF-alpha to baseline levels. Myeloperoxidase activity in all groups was no different from that of normal hearts. Mitochondrial cytochrome c release increased in both control and treatment groups. CONCLUSIONS: Bcl-2 is actively downregulated and TNF-alpha is upregulated in this model of cardiac allograft ischemia/reperfusion. Furthermore, the caspase-3 pathway is linked to this process, and blockade of caspase-3 can ameliorate reperfusion injury by upregulating bcl-2 and inhibiting TNF-alpha without affecting cytochrome c release.

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.

Zinc-mediated reduction of apoptosis in cardiac allografts.

BACKGROUND: Apoptosis is thought to occur during immune-mediated acute rejection of cardiac allografts. In vitro studies have shown that zinc inhibits the activity of the proapoptotic enzyme caspase-3. We hypothesized that ZnCl(2) would reduce acute cardiac rejection in vivo via the blockade of caspase-3-dependent apoptosis. (99m)Tc-labeled annexin V was used to measure apoptosis in cardiac allografts through nuclear imaging. Annexin V binds to phosphatidylserines, which are externalized to the outer membrane of apoptotic cells. METHODS AND RESULTS: Twenty-seven PVG rat hearts were transplanted heterotopically into the abdomen of untreated ACI rats as controls (group 1). Fifteen were scanned and euthanized on postoperative day 4, and 12 were assessed for graft survival. Group 2 and 3 rats (n=15 each) received 1 and 5 mg/kg ZnCl(2) BID IP, respectively. Nine of each of these groups were scanned and euthanized on postoperative day 4, and 6 were studied for allograft survival. Group 4 rats (n=3) received isografts. Region-of-interest analysis demonstrated a dose-dependent reduction in (99m)Tc annexin uptake in ZnCl(2)-treated allografts: 2.43+/-0.37% for group 1, 1. 97+/-0.41% for group 2, 1.21+/-0.47% for group 3, and 0.55+/-0.19% for group 4 (ANOVA, P:=0.001). Graft survival times of 6.4+/-1.7, 9. 3+/-3.0, and 11.5+/-3.4 days for groups 1, 2, and 3, respectively, were also observed (ANOVA, P:=0.001). Caspase-3 activity in the allografts showed a 3.7-fold reduction in group 3 animals compared with group 1 animals (P:=0.004). CONCLUSIONS: Apoptosis that occurs in acute cardiac allograft rejection is reduced with ZnCl(2) in a dose-dependent manner via caspase-3 inhibition.

Invasive aspergillosis in the setting of cardiac transplantation.

Among patients undergoing heart transplantation, Aspergillus is the opportunistic pathogen with the highest attributable mortality. The median time of onset from transplantation for invasive pulmonary aspergillosis (IPA) was 46 days, but the median time to first positive culture result was 104 days among patients with Aspergillus colonization but no invasive disease. Most patients with IPA presented with fever and cough within the first 90 days of transplantation and with single or multiple pulmonary nodules. None of the heart transplant recipients with either IPA or invasive extrapulmonary aspergillosis (IEPA) had associated neutropenia. Human leukocyte antigen A1 locus was found significantly more frequently among patients colonized with Aspergillus than among patients with IPA (P<.006) or IEPA (P<.001). Even in the absence of neutropenia, IPA should be suspected for heart transplant recipients who have fever and respiratory symptoms within the first 3 months of transplantation, have a positive result of culture of respiratory secretions, and have abnormal radiological findings (particularly nodules).

The bronchoalveolar lavage fluid of cystic fibrosis lung transplant recipients demonstrates increased interleukin-8 and elastase and decreased IL-10.

Cystic fibrosis (CF) patients continue to have reservoirs of Pseudomonas aeruginosa infection in their sinuses and trachea after transplantation, and studies indicate that nontransplanted CF patients have high bronchoalveolar lavage (BAL) levels of proinflammatory factors, interleukin-8 (IL-8), and elastase and decreased airway lavage levels of IL-10. The aims of our study were to measure the IL-8 and IL-10 levels and elastase activity in the BAL of lung transplant patients, with correlation to microbiologic and pathologic findings, and to identify any differences in the findings between CF and non-CF patients. Fifty serial BAL samples were collected from 38 lung transplant recipients over 8 months. The BAL supernatant fluid was cultured for bacterial, viral, and fungal organisms. Histologic tissue analysis was performed as indicated. The fluid IL-10 and IL-8 levels were measured in duplicate using ELISA techniques. Elastase activity was measured using a colorimetric assay system. The mean IL-8, IL-10, and elastase levels for the group studied were 1894 pg/ml, 394 pg/ml, and 4.2 U/ml, respectively. The CF patients had significantly higher levels of IL-8, with a mean value of 4093 pg/ml (p < 0.02), and lower IL-10, mean 217 pg/ml (n = 9). Elastase activity correlated strongly with IL-8 level (p < 0.04). Pseudomonas growth was associated with higher elastase and IL-8 concentrations (p < 0.02). There was no association between allograft rejection and the markers studied. CF transplanted patients have higher airway lavage concentrations of IL-8 and elastase correlated to the presence of Pseudomonas in the lower airway. They also have lower BAL levels of anti-inflammatory cytokine IL-10.

The utility of annual surveillance bronchoscopy in heart-lung transplant recipients.

Bronchoscopy with transbronchial biopsy (TBBx) and bronchoalveolar lavage (BAL) has an appreciable yield in detecting asymptomatic abnormalities in heart-lung transplant recipients (HLTR) during the early postoperative period. The utility of annual surveillance procedures has not been critically evaluated. We reviewed all annual bronchoscopies performed on 29 HLTR to determine the frequency of asymptomatic abnormalities. Surveillance bronchoscopies (SB) were performed on asymptomatic subjects with unchanged lung function compared with baseline. Surveillance/clinical bronchoscopies (SCB) were those performed in patients with stable decrements in lung function. Nineteen patients underwent 48 SB and 8 had 18 SCB. Five of 15 (33%) SB performed at one year yielded an abnormal TBBx (1 grade 2 acute rejection [AR], 1 grade 1 AR, 1 grade 1 AR with obliterative bronchiolitis [OB] and 2 Pneumocystis carinii pneumonia). At 2 or more years, TBBx was abnormal in 2 of 33 (6%, p = 0.024 compared with first year TBBx) (1 grade 1 AR, 1 lymphocytic bronchiolitis). Pathogens were identified in BAL in 19 (40%) SB. Fourteen (78%) SCB were abnormal. Nine (50%) revealed an abnormal TBBx (all OB), but only 2 (11%) of these altered patient management. Seven (39%) demonstrated pathogens in BAL. We conclude that in HLTR (1) surveillance TBBx rarely yields positive findings 2 or more years posttransplant, (2) surveillance TBBx seldom alters management in patients with stable decrements in lung function, and (3) BAL is useful to screen for potential pathogens.

Nuclear factor-kappaB transcription factor decoy treatment inhibits graft coronary artery disease after cardiac transplantation in rodents.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1. We hypothesized the use of ex vivo pressure-mediated delivery of transcription factor decoys (TFD) to NF-kappaB binding sites would decrease expression of adhesion molecules, and decrease reperfusion injury, acute rejection, and graft coronary artery disease (GCAD) in rat cardiac allografts. METHODS: Heterotopic heart transplants were performed on donor hearts treated with saline, 10 mg/kg LPS, 160 micromol/L NF-kappaB TFD, or 160 micromol/L scrambled sequence (NF-SC) TFD for 45 min at 78 psi (6 atm). Transfection efficiency was determined with FITC-labeled TFD. Reverse transcription-PCR and immunohistochemistry was used to analyze adhesion molecule mRNA and protein expression, respectively. Apoptosis was measured with DNA fragmentation analysis. Reperfusion injury was assessed with cardiac edema, neutrophil infiltration, and histology. Acute rejection was determined by daily palpation. Allografts were assessed at POD 90 for the development of GCAD by computer-assisted image analysis to determine intimal:medial ratio and myointimal proliferation. RESULTS: Hyperbaric pressure was an effective method of NF-kappaB TFD delivery (P<0.001 vs. controls). NF-kappaB TFD treatment led to decreased mRNA and protein expression of adhesion molecules. Treatment with NF-kappaB TFD led to a significant decrease in all reperfusion injury parameters compared to saline and NF-SC controls (P<0.01 vs. controls). Higher levels of apoptosis were seen in allografts treated with NF-kappaB TFD compared to control allografts. NF-kappaB TFD treatment prolonged allograft survival over saline and NF-SC controls (P<0.05). Myointimal proliferation and intimal:medial ratios in NF-kappaB TFD-treated allografts were significantly decreased compared to saline and NF-SC treatment (P<0.00001). CONCLUSIONS: Ex vivo pressure-mediated delivery of NF-kappaB TFD is an effective method to block adhesion molcule expression and reperfusion injury in the immediate posttransplant period. Further, NF-kappaB TFD treatment prolongs allograft survival and decreases GCAD.

Photodynamic therapy with motexafin lutetium (Lu-Tex) reduces experimental graft coronary artery disease.

BACKGROUND: Motexafin lutetium (Lu-Tex) is a photodynamic therapy (PDT) agent that localizes in atheromatous plaque in which it can be activated by far-red light. Lu-Tex biolocalization was examined in graft coronary artery disease (GCAD) with a rodent allograft model. After photoactivation, the effect on intimal proliferation was assessed. METHODS: A PVG to ACI rat heterotopic heart transplantation model was used. Lu-Tex (10 mg/kg) was intravenously administered 90 days after transplantation. Photoactivation was performed 24 hr after Lu-Tex administration. A light-emitting diode, central wavelength of 742 nm, was used to illuminate the intraperitoneally placed allografts via a laparotomy (light fluence of 75 J/cm2 at a power density of 75 mW/cm2). Animals were divided into four groups according to postoperative treatments: PDT with Lu-Tex injection and light illumination (n=21), Lu-Tex injection and laparotomy (n=14), laparotomy with light only (n=14), and laparotomy only (n=16). GCAD was quantitatively assessed 14 days after treatments. RESULTS: Lu-Tex localized in atherosclerotic plaque in vessels with GCAD. PDT significantly reduced both the percent of affected vessels and intimal proliferation compared to all other control study groups. alpha-Smooth muscle cell actin and anti-rat macrophage antibody-positive areas were significantly reduced within the neointima in allografts treated with PDT compared to all other study groups. CONCLUSIONS: PDT significantly reduced atherosclerotic lesions of GCAD. Lu-Tex-mediated PDT may, therefore, be a potential method for treating accelerated atherosclerosis associated with transplantation.

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.

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.

L-arginine polymer mediated inhibition of graft coronary artery disease after cardiac transplantation.

BACKGROUND: Nitric oxide (NO) limits the development of graft coronary artery disease (GCAD) in transplanted hearts. We hypothesized that l-arginine polymers administered to cardiac allografts ex vivo would translocate across vascular cellular membranes, up-regulate inducible nitric oxide synthase (iNOS) production of NO, and inhibit the development of GCAD. METHODS: Three groups of PVG rat donor hearts were incubated with either 0.8 ml phosphate-buffered saline, (PBS, n=12) or 50 microM L-arginine polymer solutions of length five (R5, n=12) or nine (R9, n=12) prior to heterotopic transplantation into ACI recipients. Graft vessels were scored at POD 60 and 90 for percentage luminal narrowing (%LN), intima to media ratio (I/M), and percentage affected vessels (%AV). Translocation efficiency was determined by treatment with biotinylated polymers. NO production of treated aortic segments was determined in vitro by Griess reaction. RESULTS: Translocation efficiencies were 89+/-19% (R9), 7+/-10% (R5), and 0+/-0% PBS (ANOVA, P<0.001) which corresponded to NO production in treated aortic segments of 0.175+/-0.17 (R9), 0.120+/-0.006 (R5), and 0.135+/-0.035 microM/mg (PBS), (ANOVA, P=0.002). GCAD scores at POD 60 were: %LN: 3.2+/-3.8% (R9), 12.6+/-6.7% (R5), 11.3+/-4.2% (PBS) (ANOVA, P=0.025); I/M: 0.03+/-0.04 (R9), 0.13+/-0.07 (R5), 0.12+/-0.05 (PBS) (ANOVA, P=0.037); %AV: 7+/-7% (R9), 19+/-7%(R5), 22+/-9%(PBS) (ANOVA, P=0.021). Reduction of GCAD parameters was maintained at POD 90. CONCLUSION: R9 efficiently translocated across cytoplasmic membranes, enhanced vascular NO production, and decreased neointimal hyperplasia. This ex vivo treatment may have a therapeutic role in preventing GCAD.

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.