Hyperacute rejection in the xenogenic transplanted rat liver is triggered by the complement system only in the presence of leukocytes and free radical species.

BACKGROUND: Reactive oxygen species (ROS) and nitric oxide species (NOS) are pivotal after ischemia-reperfusion. However, the role of different cells on the formation of free radical species after xenotransplantation remains elusive. We hypothesized that ROS and NOS formed during hyperacute rejection are dependent on leukocytes, erythrocytes, activated thrombocytes, and Kupffer cells (KCs). To address this issue, we developed a model of xenoperfused rat liver and assessed the relationship between free radical production and graft dysfunction. METHODS: Livers from Sprague-Dawley rats were isolated, flushed with cold Ringer solution, and perfused at physically flow rates for 120 min after 1 h of ischemia. The control group was perfused with rat whole blood (n = 9). In the study groups, the livers were perfused with human whole blood, human plasma with erythrocytes, and plasma with erythrocytes and isolated thrombocytes (n = 9/group). In an additional group, gadolinium chloride (GdCl3), a selective Kupffer cell (KC) toxic agent, was applied. Liver damage, hyperacute rejection, and the depletion of KCs were monitored histologically. Liver damage and function were determined by means of liver enzymes, portal pressure, and bile production. Malondialdehyde (MDA), nitric oxide formation, and peroxynitrite concentration, as well as total glutathione (tGSH) level, were measured as indicators for free radical formation and anti-oxidative status. RESULTS: Significant differences in the MDA, NO, peroxynitrite levels, and GSH levels after reperfusion with various cell populations were observed. Markedly high ROS/RNS production was evident in the KCs and the xenogeneic whole-blood group. The oxidative stress was mainly caused by leukocytes and to lower extent by KCs, but only in combination with leukocytes. Neither erythrocytes, thrombocytes, nor hepatocytes had an effect on the release of ROS and RNS, as we could not observe significant differences in the MDA, peroxynitrite, and NO levels in these groups compared with control. Tissue injury and hyperacute rejection were more evident in the KC and whole-blood livers. No sign of damage was observed for the control, erythrocyte, and thrombocyte group. Removal of leukocytes from the perfusate by filtration had a major protective effect on the liver function and the grade of hyperacute rejection, whereas KC depletion reduced the ROS production, but did not have an impact on the hyperacute rejection and liver damage. In all xenogeneic perfused groups, the activation of the complement was histologically observed by positive C3c and C9b. Neither KC depletion nor the removal of leukocytes or thrombocytes from the perfusate had an effect on the activation of the complement system. Damage of the rat liver by the complement system was only observed in association with leukocytes. CONCLUSION: Our data revealed that various cell populations contribute to the formation of free radicals in our model. The production of free radicals was mainly linked to leukocytes and to a minor extent to KCs, but only in combination with leukocytes. Free radicals critically contribute to injury, rejection, and dysfunction of the xenotransplanted liver. Furthermore, hyperacute rejection in the xenogeneic perfused liver is triggered by the complement system only in the presence of leukocytes and free radical formation.

Evidence of an autoregulatory mechanism of regional bone blood flow at hypotension.

BACKGROUND: Blood flow in various organs is determined by an autoregulatory mechanism that guarantees constant organ perfusion over a wide range of arterial blood pressure changes. This physiological principle has been proven for the kidney, brain and intestinal tract, but so far not for bone. This study was carried out to determine whether there is an autoregulatory mechanism of bone or not. METHODS: The fluorescent microsphere reference sample method was used to determine blood flow within the bone and kidneys. Eight anesthetized female New Zealand rabbits received left ventricular injections of fluorescent microspheres over a wide range of arterial pressure levels prior to removal of kidney, femur and tibia. Blood flow values were calculated by measurement of fluorescence intensity in kidney and bone and correlated to fluorescence intensity in the peripheral blood (reference sample). RESULTS: Despite a reduction of mean arterial pressure from 100 to 80 mmHg bone blood flow remained constant. Further reduction of mean arterial pressure results in a linear decrease in bone blood flow. CONCLUSION: The correlation between arterial pressure and organ perfusion in the bone is similar to blood flow within the kidney, indicating the presence of an autoregulated blood flow mechanism within the bone tissue.

Microcirculatory alterations after orthotopic pig-to-baboon heart transplantation.

BACKGROUND: Whilst macrohemodynamic function of porcine xenografts transplanted into baboons has been assessed perioperatively, the ability of the xenograft to maintain systemic microcirculatory perfusion has not been investigated after pig-to-baboon xenotransplantation so far. METHODS: We investigated the sublingual microcirculation of six baboons undergoing orthotopic transplantation of hCD46-transgenic pig hearts using orthogonal polarization spectral imaging. Microvascular measurements were performed after induction of anesthesia, in the early phase of cardiopulmonary bypass (CPB), during reperfusion of the porcine heart and 1 h after the xenograft had resumed its life-supporting function. Microvascular blood flow was analyzed semiquantitatively and the number of visualized cell-to-cell interactions was counted. RESULTS: The proportion of continuously perfused microvessels was 97 (96 to 97) % at baseline and 95 (94 to 97) % in the early phase of CPB. It decreased significantly (P < 0.05) during CPB to 89 (84 to 91), and alterations were still present (P < 0.05) when CPB was terminated and the xenograft had taken over systemic perfusion 83 (81 to 85) %. The microcirculatory changes correlated with the lactate levels (y = 18.1-0.18 x; r(2) = 0.55; P < 0.001), but no correlation with macrohemodynamic parameters was found. CONCLUSION: Microvascular blood flow is altered after orthotopic pig-to-baboon heart transplantation, despite systemic hemodynamic parameters being well maintained by the porcine xenograft. These changes are moderate but persist after termination of CPB. Further studies need to elucidate whether these changes are transient or add to the mortality associated with cardiac xenotransplantation.

Preclinical evaluation of a new self-expanding device for closure of muscular ventricular septal defects in a pig model.

OBJECTIVES: Aim of our study was the preclinical evaluation of a new self expanding device for interventional closure of muscular ventricular septal defects (mVSDs) in an acute pig model. BACKGROUND: Devices currently in use for closure of mVSDs still have their limitations. The deployment of the disks is dependent from the expansion of the stent, which can be associated with problems for sufficient closure of the mVSDs. This was the reason for developing a modified device with only one disk MATERIALS AND METHODS: The device was constructed in a single wire technique with a unique configured retention disk. mVSDs were created in six pigs with a specially designed punch instrument, and subsequently closed with our new device during the same session using a jugular or femoral vein approach. Potential residual shunting volumes were estimated by echocardiography and hemodynamic measurements. After closure, animals were sacrificed, and hearts were harvested for macropathologic evaluation. In two animals, MRI was performed for additional noninvasive evaluation. RESULTS: Devices were successfully implanted in all animals with good alignment of the disk to the left ventricular septum, even if the stent was oversized. Echocardiography, hemodynamics, angiography and macropathology revealed complete closure of all mVSDs. MRI and echocardiography showed a good visibility of the device. CONCLUSIONS: Our preclinical study shows successful closure of iatrogenic created mVSDs without residual shunting. The device is characterized by a more controlled deployment, an independent deployment of disk and waist, and a good alignment of the left ventricular disk to the muscular septum.

Fluorescent cardiac imaging: a novel intraoperative method for quantitative assessment of myocardial perfusion during graded coronary artery stenosis.

BACKGROUND: The purpose of the present study was to examine whether the effect of coronary stenoses of variable severity on myocardial perfusion can be quantitatively assessed in vivo by analysis of fluorescent cardiac imaging (FCI) compared with the gold standard, the fluorescent microsphere method. FCI is a novel technology to visualize coronary vessels and myocardial perfusion intraoperatively using the indocyanine green dye with an infrared-sensitive imaging device. METHODS AND RESULTS: Graded stenoses and total vessel occlusion of the left anterior descending coronary artery were created in 11 open-chest pigs. Stenoses were graded to reduce resting left anterior descending coronary artery flow by 25%, 50%, 75%, and 100% of baseline flow measured by transit-time flowmeter. FCI images were analyzed with a digital image processing system. The impairment of myocardial perfusion was quantified by background-subtracted peak fluorescence intensity and slope of fluorescence intensity obtained with FCI and compared with myocardial blood flow assessed by fluorescent microsphere. All stenoses resulted in an impairment of myocardial perfusion visualized by FCI. Occlusion of the left anterior descending coronary artery resulted in a total perfusion defect (no fluorescence intensity) of the corresponding anterior myocardial wall. During graded stenosis and total vessel occlusion, normalized background-subtracted peak fluorescence intensity and slope of fluorescence intensity decreased significantly (P<0.0001). Both background-subtracted peak fluorescence intensity (r=0.92, P<0.0001) and slope of fluorescence intensity (r=0.93, P<0.0001) analyzed by FCI demonstrated good linear correlation with fluorescent microsphere-derived myocardial blood flow. CONCLUSIONS: The impairment of myocardial perfusion in response to increased coronary stenosis severity and total vessel occlusion can be quantitatively assessed by FCI and correlates well with results obtained by fluorescent microsphere.

Reduced fibrin deposition and intravascular thrombosis in hDAF transgenic pig hearts perfused with tirofiban.

BACKGROUND: Solid organ xenograft rejection is associated with vascular injury resulting at least in part in platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis and interstitial hemorrhage. Complement activation plays a prominent role in platelet-endothelial interaction. We tested the effects of platelet GPIIb/IIIa inhibitor tirofiban during perfusion of hDAF pig hearts. METHODS: Using a working-heart model, nontransgenic and hDAF pig hearts were perfused with tirofiban or human blood only. Myocardial damage was determined by hemodynamic parameters (cardiac output, stroke work index) and creatine phosphokinase. Further monitoring included the assessment of complement factors (C3, C4), platelets, fibrinogen, ATIII, and graft histology. RESULTS: Tirofiban increased cardiac output (CO) and stroke work index (SWI) of nontransgenic pig hearts and improved superior CO and SWI of hDAF pig hearts. Although perfusion time of nontransgenic pig hearts was prolonged by tirofiban (196+/-65 min vs. 162+/-122 min), a similar effect in hDAF pig hearts (218+/-116 min vs. 222+/-30 min) could not be demonstrated. Tirofiban reduced consumption of C3 and C4 independently from hDAF. Depletion of fibrinogen was equally diminished by tirofiban and hDAF; the combination of both agents obtained no further reduction. ATIII consumption was most effectively inhibited by this combination. Intravascular fibrin deposition was reduced by tirofiban and hDAF, but particularly by the combination of the two agents. CONCLUSIONS: Improvement of heart performance and reduction of myocardial damage and intravascular thrombosis confirm a role of the GPIIb/IIIa inhibitor tirofiban for the prevention of hDAF pig heart rejection and xenograft function.

In vivo visualization of the effect of polyclonal antithymocyte globulins on the microcirculation after ischemia/reperfusion in a primate model.

BACKGROUND: Ischemia-reperfusion injury (IRI) leads to increased leukocyte adherence enhancing acute cellular rejection and microvascular dysfunction. Polyclonal antithymocyte globulins (ATGs) induce T-cell depletion and functional impairment of nondepleted lymphocytes in peripheral blood. ATGs represent an important option in the treatment of acute cellular rejection but little is known about their effects on the microcirculation in IRI. METHODS: In a perfusion system, 19 cynomolgus monkeys were used to evaluate the influence of three different ATGs on the leukocyte-endothelium interaction after cold ischemia. ATGs were administered to human blood 30 min prior to reperfusion of primate extremities. Using intravital fluorescence microscopy the postreperfusion microcirculation of skeletal muscle was visualized. RESULTS: Significant differences were found between ATG-treated and ATG-free groups concerning blood flow velocity, leukocyte count, and leukocyte-endothelium interaction. ATGs reduced microvascular leukocyte adhesion, count, and blood flow impairment. CONCLUSION: ATGs have a favorable impact on early mechanisms of IRI. Due to reduced leukocyte adherence to the antigen-presenting endothelial cells, recognition events cannot take place in the posttransplant period of reperfusion. In addition to inhibiting acute transplant rejection, increase of posttransplant blood flow supports the use of ATGs as pretransplant induction therapy.

Influence of polyclonal anti-thymocyte globulins upon ischemia-reperfusion injury in a non-human primate model.

BACKGROUND: Polyclonal anti-thymocyte globulins (ATGs) are used to induce immunosuppression and to treat acute rejection after transplantation. ATGs induce apoptosis and in peripheral T-lymphocytes having the potential to inhibit leukocyte adhesion. We analysed the influence of three different ATGs upon the microvasculature and the different cell-subpopulations after ischemia/reperfusion (IRI). MATERIALS AND METHODS: Extremities of cynomolgus monkeys were surgically isolated and flushed with Ringer's lactate at 4 degrees C. After 60 min of ischemia the limbs were reperfused with matching human blood. ATGs were added to the blood 30 min prior to the reperfusion. Four groups were generated: Tecelac-ATG group, Fresenius(S)-ATG group, Thymoglobulin-ATG group and a control group. Blood analyses were performed in blood samples taken after the beginning of the reperfusion. Biopsies from muscular tissue were obtained after the experiments. RESULTS: The number of circulating leukocytes was lower in the ATG-groups than in control. Morpho-cytological analyses showed depletion of peripheral lymphocytes. Histological examination showed less tissue damage, reduced presence of fibrin and adherent thrombocytes in the ATG-treated groups. Leukocyte infiltration, both in muscle and vascular structures, was significantly diminished in the ATG-groups in comparison to control. DISCUSSION: Our results show that ATGs have a favourable impact on early mechanisms of IRI. ATGs showed a reduction of the number of adherent leukocytes and muscle infiltrates suggesting that preoperative therapy with ATGs may have an advantageous effect on primary non-function and on chronic rejection as well as a positive influence upon IRI.

Binding of ATGs to Endothelial Cells In Vivo.

BACKGROUND Polyclonal anti-thymocyte globulins (ATGs) are immunosuppressive drugs widely used in induction of immunosuppression and treatment of acute rejection after solid organ transplantation. We have previously demonstrated that ATGs bind to endothelial cells in vitro, and are able to modulate ECs. The aim of this study was to investigate the binding of ATGs to endothelial cells under in vivo conditions. MATERIAL AND METHODS Muscle biopsies from extremities of cynomolgus monkeys were obtained after ischemia/reperfusion at 4°C. ATGs (Thymoglobulin, Sanofi-Aventis, France; 1 mg/kg) were added to the blood 30 min prior to the reperfusion. Biopsies (n=10) of patients undergoing heart transplantation and preoperatively treated with ATGs (Thymoglobulin, Sanofi-Aventis, France; 1.5 mg/kg) as induction therapy were also analyzed 6 hours and 7 days after induction. Binding of ATGs to ECs was analyzed with an anti-rabbit IgG antibody by means of immunohistochemistry. RESULTS Binding of ATGs to endothelial cells could be demonstrated in vivo in our animal experiments 4 hours after reperfusion, as well as in the clinical biopsies 6 hours after induction of immunosuppression in heart transplant patients, showing a preferred localization in post-capillary veins. No expression of ATGs on the endothelial surface could be observed after 7 days, suggesting that ATGs may be washed out from the endothelial surface in a time-dependent manner. CONCLUSIONS Our results show that ATGs are able to bind to endothelial cells in an experimental model and in clinical practice, supporting preconditioning strategies with ATGs in solid organ transplantation.

Selective pressure-regulated retroinfusion of fibroblast growth factor-2 into the coronary vein enhances regional myocardial blood flow and function in pigs with chronic myocardial ischemia.

OBJECTIVES: We sought to improve regional myocardial delivery and subsequent collateral perfusion induced by basic fibroblast growth factor-2 (FGF-2) using selective pressure-regulated retroinfusion of coronary veins for delivery. This hypothesis was tested in a newly developed pig model with percutaneous induction of chronic ischemia. BACKGROUND: Selective pressure-regulated retroinfusion of coronary veins is a catheter-based procedure that has been shown to provide effective regional delivery of drugs and gene vectors into ischemic myocardium. METHODS: A high-grade stenosis with subsequent progression to total occlusion within 28 days was induced by implanting a reduction stent graft into the left anterior descending artery (LAD). After seven days, a 30-min retroinfusion (anterior cardiac vein) was performed with (n = 7) or without (n = 7) 150 microg FGF-2 and compared with a 30-min antegrade infusion of 150 microg FGF-2 into the LAD (n = 7). Sonomicrometry to assess regional myocardial function at rest and during pacing, and microspheres to assess regional myocardial blood flow, were performed 28 days after implantation of the reduction stent. RESULTS: Retroinfusion of FGF-2 compared favorably with controls and with antegrade infusion of FGF-2 with regard to regional myocardial function at rest (18.5 +/- 4.1% vs. 5.7 +/- 2.9% vs. 7.9 +/- 1.8%, respectively, p < 0.05) and during pacing. Regional myocardial blood flow was also higher in the LAD territory after retroinfusion of FGF-2 (1.07 +/- 0.14 vs. 0.66 +/- 0.07 vs. 0.72 +/- 0.17 ml x min(-1) x g(-1), p < 0.05). CONCLUSIONS: Selective pressure-regulated retroinfusion increased tissue binding of FGF-2 and enhanced functionally relevant collateral perfusion compared with antegrade intracoronary delivery in pigs with chronic myocardial ischemia.

A new animal model to assess angiogenesis and endocrine function of parathyroid heterografts in vivo.

BACKGROUND: It is still a matter of investigation how angiogenesis and restoration of gland perfusion determine graft function after free parathyroid autotransplantation. We provide a new animal model allowing simultaneous and repetitive in vivo assessment of angiogenesis and endocrine function of parathyroid transplants. METHODS: Fresh human parathyroid tissue from patients with secondary hyperparathyroidism was grafted into dorsal skinfold chamber preparations of athymic nude mice (CD1-nu; n=8). Equivalent pieces of the same human donor specimens were heat-inactivated and served as control grafts (n=7). RESULTS: In all animals receiving parathyroid transplants, intact human parathyroid hormone levels were detectable by species-specific enzyme-linked immunosorbent assay analysis of plasma samples on day 5 after transplantation and increased by 2.5-fold over the observation period (19 days) in contrast with controls. Plasma Ca levels revealed no differences between the groups. On day 5 after transplantation, intravital fluorescence microscopy revealed murine angiogenic microvessels sprouting along nonperfused human donor vessels, and 1 week later functional microvasculature was established in all parathyroid transplants. Histologic analysis revealed well-vascularized endocrine tissue. In contrast, control grafts were necrotic and partly resorbed; they exhibited no angiogenic activity or well-vascularized fat cells indicating fatty degeneration. In addition, species-specific Western blot analysis revealed vascular endothelial growth factor expression of parathyroid transplants rather than functional vessel density as the functional parameter of angiogenesis determining transplant function in vivo. CONCLUSION: This model may serve to understand mechanisms associated with specific parathyroid transplant angiogenesis and its significance for transplant function to optimize clinical success of autotransplantation in therapy-resistant patients.

In vitro influence of polyclonal anti-thymocyte globulins on leukocyte expression of adhesion molecules.

OBJECTIVES: Polyclonal anti-thymocyte globulins (ATGs) are drugs used in the induction of immunosuppression, in the treatment of acute rejection, and in the therapy of hematologic disorders. Treatment with ATGs can produce adverse effects due to cross-reacting antibodies directed against nonmyeloid cells. This study sought to evaluate the interaction of ATGs and some adhesion molecules expressed on the surface of neutrophils and lymphocytes. MATERIALS AND METHODS: We determined the effects of different doses of 3 polyclonal ATGs on the activation and expression of lymphocyte and neutrophil adhesion molecules in whole blood by means of flow cytometry. RESULTS: ATG treatment reduced the percentage of lymphocytes gated for CD18 and CD62L, as well as the expression of CD11b, CD18, and CD62L in a dose-dependent manner. ATGs modulated the percentage of gated neutrophils for CD18. Although ATG treatment did not affect CD11b or CD62L gating in neutrophils, it did regulate expression of these adhesion markers. CONCLUSIONS: Our results show that ATGs can modify the expression levels of some of the main leukocyte adhesion molecules that are responsible for the characteristic cellular adhesion after ischemia/ reperfusion. These properties of ATGs may contribute to reduced leukocyte infiltration after solid-organ transplantation.

In vitro assessment of dose-dependent platelet activation by polyclonal antithymocyte globulins: a flow-cytometric analysis.

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in transplantation and hematologic disorders. Treatment with ATGs can induce side effects such as neutropenia and thrombocytopenia because of unspecific antibodies directed against nonmyeloid cells present in these preparations. Depletion, activation, and expression of adhesion molecules on platelets in vitro were studied in the whole blood of healthy volunteers by means of flow cytometry after incubation with different doses of three polyclonal ATGs. Our data show no ATG-mediated cytotoxic activity against platelets. ATGs are able to induce activation of platelets through increased expression of P-selectin and hLAMP-1 and higher percentages of gated thrombocytes expressing these molecules. Furthermore, increased expression of hLAMP-1 presented a dose-dependent pattern. ATGs induced activation and enhanced expression of adhesion molecules in unstimulated platelets. Increased adhesion may be responsible for undesirable side effects such as thrombocytopenia and reticulopenia.

Polyclonal antithymocyte globulins reduce the expression of IL-4 in a non-human primate model of ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a non-specific, antigen independent event, which significantly influences the outcome of transplanted organs. Interleukin-4 (IL-4) is an immunological mediator belonging to the interleukin family that mainly regulates the differentiation of T-helper lymphocytes into Th2 phenotype as well as enhances cellular activation, both are important features in IRI. The influence of polyclonal antithymocyte globulins (ATGs) on expression of IL-4 in reperfused tissues of cynomolgus monkeys (n=18) after 60 min of ischemia was assessed by immunohistochemical methods. Our results show an inhibition of the production and release of IL-4 by activated lymphocytes in the groups treated with ATGs in comparison to control, although a causal relationship between IL-4 and tissue damage was not demonstrated. Implication of IL-4 as an inflammatory mediator upon IRI must be further investigated.

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.

Adeno-associated viral vector 2.9 thymosin ß4 application attenuates rejection after heart transplantation: results of a preclinical study in the pig.

BACKGROUND: Graft survival is the most important factor for morbidity and mortality in cardiac transplantation. Improved immunosuppression significantly reduced early graft rejection. However, acute rejection may predispose to chronic rejection. Targeting both phases of the recipient's immune-reactivity by means of long-acting recombinant adeno-associated viral vectors (AAVs) encoding anti-inflammatory and cardioprotective factors appears to be a promising therapeutic approach. We investigate thymosin ß4 (Tß4) possessing anti-inflammatory and prosurvival abilities, as a means for pretransplant gene therapy. METHODS: Heterotopic, abdominal transplantation of cardiac allografts into landrace or into Munich mini pigs (n=5 per group) was performed. Transplants were transduced with AAV2.9 before transplantation by means of in situ perfusion of the donor organ. Vascuar endothelial growth factor and AAV2.9.Tß4 or AAV2.9.LacZ were added to the autologous blood used for perfusing the grafts for a period of 45 min. Immunosuppression was applied for 10 days after the operation. Transgene expression, capillary density, graft function, survival, and rejection were assessed. RESULTS: The AAV2.9 transduction induced robust overexpression of the transgene. In addition, Tß4 ameliorated inflammation, necrosis, vascular reaction (acute rejection) and in parallel improved capillary density. In addition, graft survival was significantly prolonged (10±3 days AAV2.9.LacZ vs. 31±4 days AAV2.9.Tß4). In the mini pig model, regional myocardial function of the grafts was improved by Tß4 transduction compared to LacZ (9.1%±0.9% subendocardial segment shortening in AAV2.9.LacZ vs. 15.8%±2.3% in AAV2.9.Tß4). CONCLUSION: In situ AAV2.9-mediated gene transfer of thymosin ß4 attenuated graft rejection in a heterotopic heart transplantation model. Perioperative cardioprotection by means of gene therapy might improve graft survival in cardiac allotransplantation.

Local cooling reduces regional bone blood flow.

Local cooling is very common after bone and joint surgery. Therefore the knowledge of bone blood flow during local cooling is of substantial interest. Previous studies revealed that hypothermia leads to vasoconstriction followed by decreased blood flow levels. The aim of this study was to characterize if local cooling is capable of inducing reduced blood flow in bone tissue using a stepwise-reduced temperature protocol in experimental rabbits. To examine bone blood flow we utilized the fluorescent microsphere (FM) method. In New Zealand white rabbits one randomly chosen hind limb was cooled stepwise from 32 to 2°C, whereas the contra lateral hind limb served as control. Injection of microspheres was performed after stabilization of bone and muscle temperature at each temperature level. Bones were removed, dissected and fluorescence intensity was determined to calculate blood flow values. We found that blood flow of all cooled regions decreased relative to the applied external temperature. At maximum cooling blood flow was almost completely disrupted, indicating local cooling as powerful regulatory mechanism for regional bone blood flow (RBBF). Postoperative cooling therefore may lead to strongly decreased bone blood flow values. As a result external cooling has capacity to both diminish bone healing and reduce bleeding complications.

Angiogenetic potential of Ad2/Hif-1α/VP16 after regional application in a preclinical pig model of chronic ischemia.

UNLABELLED: Hif-1α, a master regulator of ischemia-responsive gene induction, controls pro-angiogenic gene expression of VEGF-A, flt-1, IGF-1 and erythropoietin, rendering its overexpression an attractive tool for therapeutic neovascularization. Utilizing an adenoviral vector system, we investigated the efficacy of selective pressure-regulated venous retroinfusion of an enhanced Hif-1α mutant (Hif-1α/VP16) in a randomized investigator-blinded study. METHODS: Pigs were subjected to percutaneous implantation of a reduction-stent into the circumflex artery, leading to progressive stenosis and complete occlusion at day 28. Selective pressure-regulated retroinfusion of the great cardiac vein was performed at day 28 for regional delivery of either saline or empty vector or Ad2/Hif-1α/VP16. Collateral growth and global myocardial function were obtained by fluoroscopy, whereas regional blood flow and regional myocardial function were assessed by fluorescent microsphere analysis and sonomicrometry, respectively. Capillary density in the ischemic myocardium was analyzed by PECAM-1 staining. RESULTS: Compared to saline and Ad empty vector controls, overexpression of Hif-1α in the ischemic region induced an increase of small (capillary) and large (collateral) vessels, resulting in an improved perfusion of the ischemic myocardium. Concomitantly, an ischemia induced loss of myocardial function (hibernating myocardium) was resolved only after transfection with the Hif 1-α transgene, but not the empty vector or saline control. CONCLUSION: Retroinfusion of Ad2/Hif-1α/VP16, combining a master pro-angiogenic protein with regional myocardial application, may offer an efficient approach to cardiac gene therapy of chronic ischemic cardiomyopathy.

Prevention of hyperacute xenograft rejection through direct thrombin inhibition with hirudin.

BACKGROUND: Hyperacute xenograft rejection (HXR) is characterized by complement activation and intravascular thrombosis. The pathogenesis of HXR is attributed to antibodies binding to α-Gal-epitopes on the endothelial cells (EC) of the xenograft, activating complement and thrombin-mediated coagulation mechanisms. Our aim was to evaluate the influence of thrombin inhibition upon HXR and tissue integrity in an ex-vivo working heart model. MATERIAL/METHODS: Eighteen isolated porcine hearts were perfused with human whole blood in a working heart model. The blood was treated with heparin (n=9) in group G-I and with heparin and additionally recombinant hirudin (0.012 mg/ml bolus, afterwards 4.5 µg/ml/h continuously) in group G-II (n=9). The experiments were terminated at end of cardiac output. Histological analysis was performed after the experiments. RESULTS: Working heart time of G-II was significantly longer (712.0±37.8 vs. 125.0±31.4 min, p<0.01). Heart weight increase in G-II was lower (0.05±0.01 vs. 0.30±0.06%/min, p<0.01). Stroke work index and specific coronary flow improved significantly in G-II after 120 minutes. Histological analysis revealed increased tissue damage and thrombosis phenomena in G-I. Moreover, immunohistochemistry showed increased C3 and C5b-C9 upon EC of G-I. CONCLUSIONS: Direct thrombin inhibition with Hirudin could be a successful strategy in primate xenotransplantation experiments to prevent tissue damage thus improving the graft survival.

Cotransfection of vascular endothelial growth factor-A and platelet-derived growth factor-B via recombinant adeno-associated virus resolves chronic ischemic malperfusion role of vessel maturation.

OBJECTIVES: We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. BACKGROUND: Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. METHODS: In rabbits, 0.5 x 10(11) particles rAAV.VEGF-A with or without 1 x 10(12) particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 x 10(13) particles), rAAV.VEGF-A/PDGF-B (2 x 10(12) and 4 x 10(12) particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. RESULTS: In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 +/- 0.09 vs. 1.32 +/- 0.11 in rAAV.LacZ-treated limbs, p < 0.05), but not collateral growth (125 +/- 7% vs. 106 +/- 7%, p = NS) or perfusion (136 +/- 12% vs. 107 +/- 9%, p = NS). With VEGF-A/PDGF-B cotransfection, collateral growth increased to 146 +/- 9%, perfusion to 163 +/- 8% of the respective day 7 value (p < 0.05). In the pig model, retroinfusion of rAAV.VEGF-A/PDGF-B increased regional myocardial blood flow reserve from 101 +/- 4% (rAAV.Mock) to 129 +/- 8% (p < 0.05), based on collateral growth (3.2 +/- 0.3 in rAAV.Mock vs. 9.0 +/- 0.4 in rAAV.VEGF-A/PDGF-B, p < 0.05), whereas rAAV.VEGF-A did not alter flow reserve (112 +/- 7%) or collateral count (5.2 +/- 0.7). rAAV.VEGF-A/PDGF-B improved ejection fraction (55 +/- 5% vs. 34 +/- 3% in rAAV.Mock, p < 0.05) unlike rAAV.VEGF-A (37 +/- 2%). CONCLUSIONS: Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.