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.

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.

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.

Outcome in cardiac recipients of donor hearts with increased left ventricular wall thickness.

The ongoing shortage of donors for cardiac transplantation has led to a trend toward acceptance of donor hearts with some structural abnormalities including left ventricular hypertrophy. To evaluate the outcome in recipients of donor hearts with increased left ventricular wall thickness (LVWT), we retrospectively analyzed data for 157 cardiac donors and respective recipients from January 2001 to December 2004. There were 47 recipients of donor heart with increased LVWT >or=1.2 cm, which constituted the study group and 110 recipients of a donor heart with normal LVWT < 1.2 cm that formed the control group. At 3 +/- 1.5 years, recipient survival was lower (50% vs. 82%, p = 0.0053) and incidence of allograft vasculopathy was higher (50% vs. 22%, p = 0.05) in recipients of donor heart with LVWT > 1.4 cm as compared to LVWT 1.4 cm (p = 0.003), recipient preoperative ventricular assist device (VAD) support (p = 0.04) and bypass time > 150 min (p = 0.05) were predictors of reduced survival. Our results suggest careful consideration of donor hearts with echocardiographic evidence of increased LVWT in the absence of hypovolemia, because they may be associated with poorer outcomes; such hearts should potentially be reserved only for the most desperately ill recipients.

Effect of inhaled tacrolimus on cellular and humoral rejection to prevent posttransplant obliterative airway disease.

This study aimed to investigate the pharmacokinetics after tacrolimus aerosol inhalation and to assess its efficacy to suppress acute and chronic airway allograft rejection. Orthotopic tracheal transplantations were performed and tacrolimus (4 mg/kg) was administered orally (PO) or via aerosol (AER). Tracheal tissue level AUCs(0-12) were similar in both treatment groups, but blood AUCs(0-12) were approximately 5.5-fold lower with AER (p < 0.001). Interestingly, only PO animals showed elevated BUN, cholesterol and triglycerides on POD 60 (p < 0.05). Histology of grafts harvested after 6 and 60 days revealed that both treatment groups were similarly effective in suppressing graft mononuclear infiltration (p < 0.001). Cellular immune activation (assessed by IFN-gamma- and IL-4-ELISPOTS), however, was far more effectively suppressed by tacrolimus PO (p < 0.001). In both treatment groups, the vigorous alloreactive IgM-antibody surge was effectively inhibited (p < 0.001). Due to the insufficient systemic cellular immunosuppression, discontinuation of tacrolimus AER resulted in a far stronger (3.5-fold) graft infiltration on POD 8 compared to PO (p < 0.001). Tacrolimus aerosol reduces systemic side effects and effectively protects the airway graft from early cellular rejection and chronic obliterative airway disease.

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.

Wound healing complications with de novo sirolimus versus mycophenolate mofetil-based regimen in cardiac transplant recipients.

Sirolimus was introduced in de novo immunosuppression at Stanford University in view of its favorable effects on reduced rejection and cardiac allograft vasculopathy. After an apparent increase in the incidence of post-surgical wound complications as well as symptomatic pleural and pericardial effusions, we reverted to a mycophenolate mofetil (MMF)-based regimen. This retrospective study compared the outcome in heart transplant recipients on sirolimus (48 patients) with those on MMF (46 patients) in de novo immunosuppressive regimen. The incidence of any post-surgical wound complication (52% vs. 28%, p=0.019) and deep surgical wound complication (35% vs. 13%, p=0.012) was significantly higher in patients on sirolimus than on MMF. More patients on sirolimus also had symptomatic pleural (p=0.035) and large pericardial effusions (p=0.033) requiring intervention. Logistic regression analysis showed sirolimus (p=0.027) and longer cardiac bypass time (OR=1.011; p=0.048) as risk factors for any wound complication. Sirolimus in de novo immunosuppression after cardiac transplantation was associated with a significant increase in the incidence of post-surgical wound healing complications as well as symptomatic pleural and pericardial effusions.

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).

Regulation of matrix metalloproteinases and effect of MMP-inhibition in heart transplant related reperfusion injury.

PURPOSE: Metalloproteinases (MMPs) regulate extracellular matrix turnover and degrade basal lamina. Aim of the study was to examine the regulation of MMPs and the effect of an MMP inhibitor in transplant related ischemia/reperfusion injury. METHODS: Heterotopic cardiac transplantation was performed after 4 h of ischemia in three groups of six rats: allografts (black hooded inbred strain, PVG donor/August Copenhagen Irish inbred strain recipient); allografts treated with a competitive MMP-inhibitor (Batimastat) 15 mg/kg every 24 h; isografts (PVG donor and recipient). Normal PVG hearts served as a control. Hearts were explanted after 72 h of reperfusion. Expression of MMP-2 and -9 was measured using gelatin zymography. Proteolytic activity was measured using a gelatinase activity assay. Myeloperoxidase activity and tumor necrosis factor-alpha (TNF-alpha) were measured as markers of inflammatory response. Immunostaining for collagen IV and laminin was used to study degradation of basal lamina. RESULTS: There was a significant increase of MMP-2 expression in allografts (2271+/-571 microg/ml) as compared to normal (683+/-139 microg/ml) and the Batimastat-treated (259+/-140 microg/ml, P<0.05) groups. Although pro-MMP-2 expression was equally high in the untreated iso- and allograft group (75+/-23 versus 62+/-30 microg/ml) MMP-2 expression in the isograft hearts was significantly lower (359+/-267 microg/ml) suggesting activation of the pro-form by an immunologic mechanism. Pro-MMP-9 levels were significantly higher in the untreated iso- and allograft groups as opposed to normal hearts and MMP-inhibited hearts. MMP-9 was completely inhibited by Batimastat treatment. Collagenolytic activity was lower in the treated group as compared to untreated allografts (538+/-140 versus 384+/-97 microg/ml, P<0.05), demonstrating effective inhibition of MMPs by Batimastat. In the treated group a lesser extent of basement membrane component alterations could be demonstrated by laminin and collagen IV staining. There was a significant reduction in myeloperoxidase activity (P=0.027) as well as lower TNF-alpha levels (ns) in the in the Batimastat treated group. CONCLUSION: Ischemia leads to an increase in MMP expression and degradation of basal lamina. This process is enhanced in allografts as compared to isografts suggesting additional activation of MMPs by immunologic mechanisms. MMP-inhibition is effective in preventing the proteolytic activity of MMPs and may alter the host rejection response by preserving extracellular matrix components and basement membranes.

Hamster cardiac xenografts are protected against antibody mediated damage, early after transplantation to Lewis rats.

Antibodies play a crucial role in the rejection of xenografts. We tested the hypothesis that xenografts are protected against antibody-mediated attack early after transplantation in a concordant model. We investigated the role of xenoreactive antibodies as a stimulus for protection and the effects of a total blockade of the antibody response by the leflunomide analog malononitrilamide 279. Hamster cardiac xenografts were transplanted to Lewis rat recipients. Second transplants and retransplants of xenografts were performed to untreated rats that had a xenograft in place for 3 d. Untreated rats rejected hamster cardiac xenografts after 4.0 +/- 0.0 d. Significant levels of anti-donor IgM, as measured by flowcytometry, were present on day 3 after transplantation (11.2% +/- 2.8 vs. 1.2% +/- 0.0 on day 0, P < 0.001). 'Fresh' second xenografts transplanted to rats that had a first xenograft in place for 3 d and had anti-hamster antibodies, underwent hyperacute rejection. The first xenografts remained functioning. Xenografts that were removed on day 3 from untreated rats and then retransplanted remained functioning. Xenografts that were removed on d 3 from rats that had been treated with malononitrilamide 279, 15 mg/kg/d and were retransplanted underwent hyperacute rejection. IgM levels at the time of removal were 1.1% +/- 0.5 in these rats and not different from baseline (P = 0.96). We conclude that xenografts are protected against antibody-mediated damage early after transplantation. The presence of anti-donor antibodies might be an essential stimulus for the induction of protection. There seems to be a delicate balance between the injurious and protective effects of antibodies. Treatment strategies that are designed to block antibody formation completely might prevent the induction of protection.

Pre-transplant blood transfusion induces tolerance to hamster cardiac xenografts in athymic nude rats.

The effects of pre-transplant blood transfusion vary from induction of antibodies and accelerated graft rejection, to prolonged survival and even tolerance. The beneficial 'transfusion effect' in allotransplantation is believed to be merely T-cell mediated. In xenotransplantation, T-cell independent mechanisms form a major hurdle. In this study we investigated the effects of pre-transplant hamster blood transfusion on the survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. Nude rats rejected xenografts after 3.8 +/- 0.5 d (n = 8), and immunocompetent Lewis rats after 4.0 +/- 0.5 d (n = 8), following a similar IgM response (P = NS). Hamster blood transfusion 3 d before transplantation in nude rats led to an IgM response and long-term xenograft survival in 17/20. Timing was of importance: blood transfusion 7 d before transplantation resulted in 45% long-term xenograft survival (n = 20). Injection of purified hamster erythrocytes, leukocytes or minced heart also led to survival of xenografts for > 100 d in nude rats, but not in all cases. Second xenografts transplanted to long-term survivors did not provoke an IigM response, and were accepted for > 100 d (n = 4). Transfer of serum from long-term survivors to untreated nude rats resulted in survival of xenografts for > 100 d (n = 4). In Lewis rats pre-transplant blood transfusion induced hyperacute rejection of xenografts after 158 +/- 128 min (n = 8, P < 0.01). We conclude that pre-transplant hamster blood transfusion can induce long-term survival of hamster cardiac xenografts in T-cell deficient athymic nude rats. This blood transfusion effect is mediated by humoral factors and can be transferred by serum. Elucidation of underlying mechanisms might provide some insight into xenotransplantation nonresponsiveness of T-cell independent immunefactors.

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.

Rabbit antithymocyte globulin versus OKT3 induction therapy after heart-lung and lung transplantation: effect on survival, rejection, infection, and obliterative bronchiolitis.

The superiority of different induction therapies after heart-lung and lung transplantation is not clearly established; specifically, whether monoclonal (OKT3) or polyclonal antibody induction therapy provides any advantage. Between 1989 and 1991 we used induction therapy with either rabbit antithymocyte globulin (RATG) or OKT3, given at random based on the availability of RATG. RATG was used in 25 patients (RATG group 1) and OKT3 in 38 patients (OKT3 group 1). Early results suggested a survival advantage with RATG. From 1992 until 1997 we used RATG induction therapy in 108 patients (RATG group 2). This study analyzed longer-term survival, infection, rejection, and obliterative bronchiolitis (OB) rates for RATG group 1 and OKT3 group 1 and assessed outcomes for RATG group 2. The 1-, 3-, and 5-year survival for RATG group 1 was 72 %, 72 %, and 52 % and for OKT3 group 1 was 63 %, 49 %, and 34 % (P < 0.05). The 1- and 3-year survival for RATG group 2 was 84 % and 74 %. The 1-, 3-, and 5-year actuarial freedom rates from lung rejection for RATG group 1 were 38 %, 38 %, and 31 % and for OKT3 group 1 were 21 %, 0 %, and 0 % (P < 0.01). The linearized rate (events/100 patient days) of all infections at 3 months was 1.55 +/- 0.28 for RATG group 1 and 2.19 +/- 0.27 for OKT3 group 1 (P = NS). The infection rate for RATG group 2 was 1.60 +/- 0.13. The actuarial rates of freedom from OB at 1, 3, and 5 years for RATG group 1 were 84 %, 51 %, and 45 % and for OKT3 group 1 were 77 %, 61 %, and 36 % (P = NS), while for RATG group 2 the rates were 97 % and 92 % at 1 and 3 years (P < 0.01 vs RATG group 1 and OKT3 group 1). The use of RATG induction therapy from 1989 through 1991 resulted in improved actuarial survival and less rejection, without increased infection rates. The use of RATG since 1992 has continued to result in similar outcomes for survival, infection, and rejection. The time to onset of OB has improved further in recent years. This may be a result of recent improvements in cytomegalovirus (CMV) prophylaxis.

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.

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.

Severe tricuspid regurgitation after heart transplantation.

BACKGROUND: Tricuspid regurgitation (TR) is common after heart transplantation. However, the incidence of severe TR and the incidence of symptoms after echocardiographic diagnosis of severe TR have not been documented. The purpose of this study is to determine the incidence of severe TR and its clinical significance in the heart transplant population. METHODS: We reviewed echocardiograms (echo) of all heart transplant patients coming for regular echocardiographic follow-up between 1990 and 1995. We reviewed the charts of all patients who had echo diagnosis of severe TR. RESULTS: A total of 336 patients had echo follow-up during this time period. The number of months post-heart transplant to last echo was 54 +/- 50 (range, 1 to 265 months). Ninety patients had moderate TR and 23 patients had severe TR. Mean time from heart transplantation to diagnosis of severe TR was 43 +/- 38 months (range, 1 to 132). Using Cutler-Ederer analysis, at 5 years, 92.2% of surviving patients were free from severe TR. At 10 years, 85.8% of surviving patients were free from severe TR. Of the 23 patients with severe TR, 17 had charts available for review. The mean number of prior endomyocardial biopsies was 28 +/- 21 (range, 3 to 88). These patients were followed for 35 +/- 18 months after diagnosis. During this period, they developed significant heart failure and peripheral edema. Six patients eventually underwent tricuspid valve replacement. CONCLUSIONS: Moderate to severe TR commonly occurs following heart transplantation. Severe TR is associated with significant morbidity.