Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles.

Muscle precursor cell (myoblasts) transplantation is considered as a potential approach to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. The study purpose was to verify the implication of hypoxia in the myoblast death observed after their transplantation and also to evaluate the potential beneficial effects of vascular endothelial growth factor (VEGF) overexpression on myoblast engraftment in a murine model. Pimonidazole hydrochloride (hypoxyprobe-1) was used to mark selectively myoblasts to evaluate their hypoxia in vivo. In vitro, hypoxia was induced by culturing human myoblasts in hypoxic environment. In vitro effects of VEGF(165) on survival of human cells was assessed by Hoescht-PI labeling. Tibialis anterior (TA) female mouse muscles were electroporated with a plasmid containing the VEGF(165) or with an empty vector. Circulating VEGF concentration was assessed by ELISA. After 2 weeks of electroporation, severe combined immunodeficient (SCID) mice were transplanted with 800 000 human male myoblasts labeled with radioactive thymidine. Mouse muscles were harvested 2 and 4 days later and myoblast survival and proliferation were evaluated by scintigraphy and Y chromosome quantitative PCR. The long-term graft success was evaluated using gamma-radiograph imaging and by counting the dystrophin positive muscle fibers. Hypoxyprobe labeling has shown that most of the transplanted myoblasts were hypoxic. The transplantation of radioactive male myoblasts in female mice electroporated with the VEGF(165) plasmid demonstrated that VEGF reduced their death by 10% but did not improve their proliferation. VEGF(165) enhanced human myoblast survival in vitro under hypoxic conditions. Electroporation of TA muscles of SCID mouse with the vector coding for VEGF(165) promoted angiogenesis and improved by 1.5-fold the success of myoblast transplantation in comparison with the control mice that were electroporated with the empty vector. These results indicate that hypoxia is partially responsible for the death of the transplanted myoblasts. VEGF can be used to improve myoblast survival and the graft success.

Membrane lipid acyl group alterations in cells infected with a temperature-conditional mutant of Rous sarcoma virus.

The increased percentage of oleic acid and decreased percentage of arachidonic acid which occurs in the lipids of chicken embryo fibroblasts transformed by Rous sarcoma virus was shown to be transformation specific rather than a consequence of virus infection. Cells infected with a temperature conditional mutant of Rous sarcoma virus (RSV-T5) had a normal type fatty acid composition when held at the restrictive temperature of 41 degrees C, but had a transformed type fatty acid composition when held at the permissive temperature of 36 degrees C. However, these acyl group changes occurred slowly in the course of transformation, suggesting that they are not a primary event in the genesis of the transformed phenotype.

Enhanced myocardial angiogenesis by gene transfer with transplanted cells.

UNLABELLED: BACKGROUND The combination of myocardial cell transplantation and angiogenic gene transfer may improve postinfarction left ventricular (LV) perfusion. We evaluated the angiogenic effect of heart cells transfected with vascular endothelial growth factor (VEGF) and transplanted into a myocardial scar. METHODS AND RESULTS: Donor rat heart cells were transfected with plasmids encoding VEGF(165) and green fluorescence protein. Syngeneic adult rats underwent LV cryoinjury to create a transmural scar. Three weeks later, 4x10(6) transfected heart cells (n=14), untransfected heart cells (n=13), or culture medium (n=16) were transplanted into the center of the scar. After 5 weeks, LV function, quantitative histology, and regional blood flow were evaluated. Plates of heart cells transfected with VEGF(165) produced 6.1 times more intracellular VEGF than nontransfected cells. Capillary density (mean+/-SEM) per high-power field in the center of the myocardial scar was 1.1+/-0.02 in control rats, 3.9+/-0.11 in untransfected rats, and 6.3+/-0.11 in transfected rats (P=0.0002). Capillary density in the border zone around the scar was 1.9+/-0.03 in control rats, 6.4+/-0.10 in untransfected rats, and 8.7+/-0.16 in transfected rats (P=0.004). Regional blood flow within the scar was 8.8+/-0.8% of normalized flow in control hearts, 10.4+/-0.7% in hearts transplanted with untransfected cells, but 17.6+/-1.2% in hearts transplanted with transfected cells (P=0.03 versus control, P=0.07 versus nontransfected). There was no difference in LV function attributable to transplantation with transfected cells at the time point studied. CONCLUSIONS: Transplantation of heart cells transfected with VEGF induced greater angiogenesis than transplantation of unmodified cells. Combined gene transfer and cell transplantation strategies may improve postinfarction LV perfusion and function.

Heart cell transplantation improves heart function in dilated cardiomyopathic hamsters.

BACKGROUND: Little is known about the effect of heart cell transplantation into the dilated cardiomyopathic myocardium. This study was designed to evaluate the effect of heart cell transplantation into dilated cardiomyopathic hamsters. METHODS AND RESULTS: Ventricular heart cells were isolated from 4-week-old BIO 53. 58 hamsters and cultured for 2 weeks before transplantation. The cells were labeled with bromodeoxyuridine (BrdU) before transplantation for identification. Adult hamsters (17 weeks old) were used as recipients. Heart cells (4 x 10(6) cells) or culture medium was transplanted into the left ventricular free wall (transplantation and control groups, respectively, n=12 each). Sham-operated hamsters (n=12) underwent the surgery but not the transplantation. Cyclosporine A was administered subcutaneously to all hamsters daily after the operation. Four weeks after the transplantation, heart function was evaluated with the use of a Langendorff preparation. Histology showed severe focal myocardial necrosis in all groups. BrdU-stained tissue was found at the cell transplantation sites. The transplanted hearts had greater (P:<0. 001) developed pressures at all balloon volumes and improved dP/dt (transplantation 915+/-253 versus control 453+/-120 and sham 530+/-187 mm Hg/s, P:<0.001, balloon volume of 15 microL). No differences in ventricular function were found between control and sham-operated hamsters. CONCLUSIONS: The transplanted ventricular heart cells formed cardiac-like tissue in cardiomyopathic myocardium and improved its contractile function.

Mutagenicity and cytotoxicity of malonaldehyde in mammalian cells.

Malonaldehyde (MA), a lipid peroxidation product derived from polyunsaturated fatty acids, is cytotoxic to a murine L5178Y lymphoma cell line cultured in vitro. Exposure of cells for 24 hours to as little as 20 microM MA produced detectable cytotoxicity as well as an increased number of mutants among survivors, using thymidine or methotrexate resistance as genetic markers. The induced mutation frequency, within the range of MA concentrations tested (10-100 microM), is dose-dependent. Significant division delay, which results in unbalanced growth, is also observed in MA-treated cells. It is suggested that MA crosslinks with DNA and mutagenizes cells through the error-prone repair system. In order to relate the degree of mutagenicity of MA in reference to other mutagenic agents in mammalian cells, the mutation frequency of the thymidine-resistance marker in L5178Y induced by X-irradiation is also presented. The significance of lipid peroxidation in relation to carcinogenesis and the various theories of aging will be briefly discussed.

The changing pattern of reoperative coronary surgery: trends in 1230 consecutive reoperations.

OBJECTIVE: We noted an increasing risk profile of patients undergoing reoperative coronary surgery. We evaluated the risk compared with primary procedures, our results over a 16-year span, and the predictors of hospital outcomes after redo surgery. METHODS: We analyzed 20,614 patients undergoing isolated coronary surgery at our institution from 1982 to 1997. Of these, 1230 (6.0%) were undergoing reoperation. Independent predictors of outcomes were identified by multivariable regression. RESULTS: The prevalence of reoperation peaked in 1994 at 8.2%. Patients undergoing reoperation were more likely to be male, to have left ventricular dysfunction and worse symptoms, and to require an urgent operation than patients undergoing a primary operation (P <.0001). Perioperative myocardial infarctions (3.7% vs 7.4%), low-output syndrome (9.0% vs 24.0%), and death (2.4% vs 6.8%) were more common in patients undergoing reoperation (all P <.0001). Over the years, the risk profile of patients undergoing reoperation increased. Age, left ventricular dysfunction, severity of symptoms, extent of coronary artery disease, left main stenosis, and requirement for urgent or emergency operations increased with time (P <.05). However, mortality, myocardial infarction, and low-output syndrome have remained constant. The independent predictors of mortality after reoperative surgery were increased age, greater Canadian Cardiovascular Society symptom class, earlier year of operation, and greater left ventricular dysfunction. After 1990, analysis of an expanded data set also identified peripheral vascular disease and failure to use retrograde cardioplegia as predictors of mortality. CONCLUSIONS: Improving results of reoperative surgery have been offset by an increasing patient risk profile. Meticulous operative technique and retrograde cardioplegia may permit good results in these high-risk patients.

Transplantation of cryopreserved cardiomyocytes.

BACKGROUND: The present study examined the survival and rate of contraction of (1) cardiomyocytes cultured from cryopreserved fetal rat myocardium and (2) cryopreserved cultured cardiomyocytes. In addition, the effects of transplantation of cryopreserved fetal cardiomyocytes were evaluated. METHODS: Segments of fetal rat myocardial tissue (0.2, 2.0, and 6.0 mm(3) mince size) and cultured cardiomyocytes were cryopreserved in liquid nitrogen for 1, 2, and 4 weeks. After cryopreservation, the tissue samples and cultured cardiomyocytes were thawed at 37 degrees C and cultured, and cell proliferation and rate of contraction were determined. Cultured cryopreserved (n = 5) and noncryopreserved (control, n = 5) fetal cardiomyocytes were transplanted into the subcutaneous tissue and into a transmural left ventricular free wall scar of Sprague-Dawley rats (n = 3). The survival and rate of contraction of these transplanted cells were also examined. RESULTS: Cryopreservation of cultured fetal cardiomyocytes resulted in viable and functional cardiomyocytes although the cell number and percentage of beating cells were diminished. Survival of cardiomyocytes isolated from cryopreserved fetal myocardium was a function of tissue size before cryopreservation; the lowest survival was recorded in tissues with the largest mince size (6.0 mm(3)). The subcutaneous transplants contracted spontaneously and regularly with an idioventricular rhythm. In addition, the transplanted cardiomyocytes were elongated and formed a myocardium-like pattern with blood vessels present within the contractile tissue. In the transmural left ventricular scar, both control and experimental fetal cardiomyocyte transplants formed myocardium-like tissue. CONCLUSIONS: The present study uncovers the following key observations: (1) cryopreservation of fetal cardiomyocytes and cardiomyocytes isolated from cryopreserved myocardial tissue results in viable and functional cells, (2) cryopreserved fetal cardiomyocytes can be successfully transplanted into subcutaneous and myocardial scar tissue, and (3) improvements in cryopreservation techniques are required to augment the rates of cardiomyocyte survival observed in the study.

Mitral valve repair and replacement for rheumatic disease.

OBJECTIVES: Mitral valve repair may be technically feasible in patients with suitable anatomy, but the appropriateness of repair for rheumatic disease remains controversial. We evaluated our late outcomes after mitral repair and replacement for rheumatic disease. METHODS: Five hundred seventy-three patients underwent mitral valve surgery for rheumatic disease at our institution from 1978-1995. Follow-up was 98% complete (mean, 68 +/- 46 months). Survival and morbidity were evaluated by Kaplan-Meier analysis and Cox regression, including propensity score analysis. RESULTS: Mean age was 54 +/- 14 years, 55% of patients had congestive heart failure, 22% were undergoing redo mitral valve surgery, and 9% also underwent coronary bypass. Mitral stenosis was present in 53%, regurgitation in 15%, and both in 32%. Valve repair was performed in 25%, bioprosthetic replacement was performed in 28%, and a mechanical valve was placed in 47%. Patients undergoing repair were younger and less likely to be undergoing reoperation or to have atrial fibrillation than those undergoing replacement (P =.001). The operative mortality rate was 4. 2%. Better late cardiac survival was independently predicted by valve repair rather than replacement (P =.04) after adjustment for baseline differences between patients. Freedom from reoperation was greatest (P =.005) but that from thromboembolic complications was worst (P <.0001) after mechanical valve replacement. Twenty-three patients underwent reoperation after initial repair, with no operative deaths. CONCLUSIONS: Mechanical valves minimize reoperation but limit survival and increase thromboembolic complications. Patients undergoing valve repair had improved late cardiac survival independent of their preoperative characteristics. Rheumatic mitral valves should be repaired when technically feasible, accepting a risk of reoperation, to maximize survival and reduce morbidity.

Predictors of operative risk for coronary bypass operations in patients with left ventricular dysfunction.

OBJECTIVES: The prevalence of ventricular dysfunction in patients undergoing coronary operations, as well as the prevalence of other risk factors in these patients, has been increasing. We identified the predictors of mortality and morbidity in patients with ventricular dysfunction to permit more accurate evaluation of risk and to direct future strategies to improve outcomes. METHODS: Demographic, intraoperative, and outcome data were collected prospectively on 20,614 patients undergoing isolated coronary operations at our institution from 1982-1997. Multivariable regression analyses were used to identify the independent predictors of mortality and low-output syndrome. RESULTS: Moderate ventricular dysfunction (ejection fraction, 20%-40%) was noted in 4107 (19.9%) patients, and severe dysfunction (ejection fraction, <20%) was noted in 680 (3.3%) patients. Patients with worse ventricular function had an increasing prevalence of other risk factors with time. Mortality decreased between the 1982-1986 and 1987-1991 cohorts but did not decrease further. Low-output syndrome was less common in the 1992-1997 cohort than in previous years. The predictors of mortality were ventricular dysfunction, age, reoperation, year of operation, urgency, female sex, and left main stenosis. Low-output syndrome was predicted by ventricular dysfunction, reoperation, year of operation, female sex, urgency, extensive coronary disease, age, left main stenosis, and symptom class. CONCLUSIONS: Despite the increasing prevalence and risk profile of patients with ventricular dysfunction, mortality rates and incidence of low-output syndrome declined with time. Patients with severe dysfunction were at greatest risk when facing reoperation or urgent operation. Earlier intervention and more aggressive preoperative optimization may improve outcomes in these high-risk patients.

Reoperative coronary bypass surgery: effect of patent grafts and retrograde cardioplegia.

OBJECTIVE: To determine the effects of patent or diseased aorta-coronary bypass grafts and retrograde cardioplegia on mortality during reoperative coronary bypass surgery. METHODS: We conducted a retrospective review of prospectively gathered data, supplemented by systematic chart review, of all patients (n = 744) undergoing reoperative coronary bypass surgery at our institution between 1990 and 1997. Independent predictors of survival were determined by stepwise logistic regression analysis. RESULTS: At least one patent or stenosed graft to the left anterior descending artery was present in 50% of patients, to the circumflex territory in 27% of patients, and to the right coronary artery territory in 33% of patients. The previous left anterior descending graft was a saphenous vein in 82% and a left internal thoracic artery in 18% of patients. In-hospital mortality occurred in 42 (5.6%) patients. Patent or diseased grafts of any coronary artery territory did not significantly increase the risk of mortality. Retrograde cardioplegia use increased in more recent years, was more frequent in patients with stenosed grafts, and was associated with improved survival. Independent predictors of mortality were as follows (with odds ratios and 95% confidence intervals in parentheses): failure to use retrograde cardioplegia (odds ratio 2.81; 1.28-6.20), New York Heart Association class (odds ratio 2.69; 1.25-5.81), peripheral vascular disease (odds ratio 2.60; 1.25-5.41), and left ventricular grade (2.07; 1.31-3.27). CONCLUSIONS: In this series, patent or stenosed grafts were not associated with an increased risk of mortality during reoperative coronary bypass surgery, possibly because of increased use of retrograde cardioplegia in this patient group. We strongly recommend the routine use of retrograde cardioplegia during redo coronary bypass surgery.

Ventricular function after normothermic versus hypothermic cardioplegia.

Warm cardioplegia produced by essentially continuous infusion has been used as an alternative to traditional cold intermittent infusion techniques during cardiac surgery, but its effects on postoperative left ventricular function have not been defined. We performed a randomized clinical trial to assess the effects of warm and cold blood cardioplegia on load-independent indices of ventricular function. Fifty-three patients were randomized to warm (n = 27) or cold (n = 26) cardioplegia. Myocardial oxygen consumption, lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during cardioplegia and reperfusion. In 13 patients per group, pressure-volume loops were constructed and ventricular function was assessed 3 hours after the operation. Warm cardioplegia resulted in greater myocardial lactate production but improved recovery of oxygen consumption during reperfusion. Depletion of adenosine triphosphate was similar between groups, but total adenine nucleotides (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate) fell further during warm cardioplegia. Cold cardioplegia was associated with an accumulation of adenosine diphosphate and adenosine monophosphate. Creatine kinase MB isoenzyme release was reduced in the warm group. Three hours after the operation, end-systolic elastance and preload-recruitable stroke work index were increased after warm cardioplegia, and early diastolic relaxation was also increased. Increased systolic function after warm cardioplegia may have been related to improved myocardial protection, elevated arterial lactate concentrations, or increased circulating catecholamine levels. Altered diastolic compliance in the warm group may reflect greater active relaxation during early diastolic filling.

Vitamin E for coronary bypass operations. A prospective, double-blind, randomized trial.

BACKGROUND: Free radical lipid peroxidation contributes to the abnormal metabolism and ventricular function frequently seen after cardiac operations. Antioxidants may improve metabolic and functional recovery. METHODS: A prospective, randomized, double-blind clinical trial was conducted to determine the effects of vitamin E (alpha-tocopherol) (n = 14) or a corn oil placebo (n = 14) in patients undergoing elective coronary bypass operations. The RRR-alpha-tocopheryl acetate doubled the alpha-tocopherol levels in the heart. Myocardial metabolism and ventricular function were assessed after the operation. RESULTS: Atrial pacing induced myocardial lactate production in the control patients but lactate consumption in the alpha-tocopherol-treated patients on bypass 25 minutes after crossclamp release. Left ventricular stroke work indices were higher, at similar ventricular volumes, in the alpha-tocopherol-treated group, which indicates improved preload recruitable stroke work, and diastolic compliance was greater 4 hours after the operation. The postoperative creatine kinase cardiac isoenzyme levels were lower in the patients who received alpha-tocopherol. CONCLUSIONS: Pretreatment with alpha-tocopherol sufficient to double the myocardial concentrations had a small but significant metabolic and functional effect after elective coronary bypass operations when compared with placebo. These results do not justify pretreatment of low-risk patients, but they do justify an evaluation in high-risk patients.

The effect of warm heart surgery on postoperative bleeding.

The effects of normothermic systemic perfusion (35 degrees to 37 degrees C; n = 73) were compared with those of moderately hypothermic systemic perfusion (25 degrees to 29 degrees C; n = 73) with respect to blood loss, transfusion requirements, and platelet levels in 146 patients undergoing isolated, primary coronary artery bypass grafting. In addition, most patients were given an antifibrinolytic medication during operation as follows: tranexamic acid (10 gm intravenously; n = 63), epsilon-aminocaproic acid (15 gm intravenously; n = 63), or no drug as a control. (n = 20). Normothermic patients tended to bleed less at 24 hours (warm, 864 +/- 42 ml and cold, 918 +/- 68 ml), but these differences were not statistically significant. Patients receiving either tranexamic acid or epsilon-aminocaproic acid, regardless of perfusion temperature, bled less after 6, 12, and 24 hours than did cold control patients (p less than 0.05). Warm control patients also bled less than did cold control patients after 6 or 12 hours (p less than 0.05), and neither drug further reduced blood loss in these patients. Circulating platelet levels were better preserved in patients receiving either tranexamic acid or epsilon-aminocaproic acid and in patients with warm perfusion and no drug than in cold control patients. Normothermic systemic perfusion, tranexamic acid, and epsilon-aminocaproic acid each reduced postoperative blood loss and preserved platelets.

Insulin cardioplegia for elective coronary bypass surgery.

BACKGROUND: Improved methods of myocardial preservation are required to reduce the morbidity and mortality of coronary bypass surgery for high-risk subgroups. Metabolic stimulation with insulin, glucose solutions, or both has been proposed as a method to preserve the ischemic myocardium. We performed a prospective, double-blind, randomized trial to evaluate the effects of insulin and glucose as cardioplegic additives when used as part of a tepid continuous blood cardioplegic strategy. METHODS: We randomized 56 male patients undergoing elective isolated coronary bypass surgery to 1 of 4 cardioplegic groups containing either 42 or 84 mmol/L glucose with or without 10 IU/L of insulin. Perioperative assessments of myocardial metabolism and left ventricular function were performed. RESULTS: Insulin-enhanced cardioplegia was associated with beneficial effects on both myocardial metabolic and functional recovery after cardioplegic arrest. Insulin's effect was independent of the ambient glucose concentration. CONCLUSIONS: Cardioplegic formulations containing a 42 mmol/L concentration of glucose and a 10 IU/L concentration of insulin provide significant benefit to patients undergoing isolated coronary bypass surgery. The clinical effect of these formulations will need to be assessed in high-risk subgroups of patients, such as those with unstable angina, recent myocardial infarction, or poor left ventricular function.

Construction of a bioengineered cardiac graft.

OBJECTIVES: Currently available graft materials for repair of congenital heart defects cause significant morbidity and mortality because of their lack of growth potential. An autologous cell-seeded graft may improve patient outcomes. We report our initial experience with the construction of a biodegradable graft seeded with cultured rat or human cells and identify their 3-dimensional growth characteristics. METHODS: Fetal rat ventricular cardiomyocytes, stomach smooth muscle cells, skin fibroblasts, and adult human atrial and ventricular cardiomyocytes were isolated and cultured in vitro. These cells were injected into or laid onto biodegradable gelatin meshes, and their rate of proliferation and spatial location within the mesh was evaluated by using a cell counter and histologic analysis. RESULTS: Rat cardiomyocytes, smooth muscle cells, and fibroblasts demonstrated steady proliferation over 3 to 4 weeks. The gelatin mesh was slowly degraded, but this process was most rapid after seeding with fibroblasts. Human atrial cardiomyocytes proliferated within the gelatin meshes but at a slower rate than that of fetal rat cardiomyocytes. Human ventricular cardiomyocytes survived within the gelatin mesh matrix but did not increase in number during the 2-week duration of evaluation. Grafts seeded with rat ventricular cells exhibited spontaneous rhythmic contractility. All cell types preferentially migrated to the uppermost surface of each graft and formed a 300- to 500-microm thick layer. CONCLUSIONS: Fetal rat ventricular cardiomyocytes, gastric smooth muscle cells, skin fibroblasts, and adult human atrial cardiomyocytes can grow in a 3-dimensional pattern within a biodegradable gelatin mesh. Similar autologous cell-seeded constructs may eventually be applied to repair congenital heart defects.

Fetal cell transplantation: a comparison of three cell types.

OBJECTIVE: We have previously reported that fetal cardiomyocyte transplantation into myocardial scar improves heart function. The mechanism by which this occurs, however, has not been elucidated. To investigate possible mechanisms by which cell transplantation may improve heart function, we compared cardiac function after transplantation of 3 different fetal cell types: cardiomyocytes, smooth muscle cells (nonstriated muscle cells), and fibroblasts (noncontractile cells). METHODS: A left ventricular scar was created by cryoinjury in adult rats. Four weeks after injury, cultured fetal ventricular cardiomyocytes (n = 13), enteric smooth muscle cells (n = 10), skin fibroblasts (n = 10), or culture medium (control, n = 15 total) were injected into the myocardial scar. All rats received cyclosporine A (INN: ciclosporin). Four weeks after transplantation, left ventricular function was evaluated in a Langendorff preparation. RESULTS: The implanted cells were identified histologically. All transplanted cell types formed tissue within the myocardial scar. At an end-diastolic volume of 0.2 mL, developed pressures in cardiomyocytes group were significantly greater than smooth muscle cells and skin fibroblasts groups (cardiomyocytes, 134% +/- 22% of control; smooth muscle cells, 108% +/- 14% of control; skin fibroblasts, 106% +/- 17% of control; P =.0001), as were +dP/dt(max) (cardiomyocytes, 119% +/- 37% of control; smooth muscle cells, 98% +/- 18% of control; skin fibroblasts, 92% +/- 11% of control; P =. 0001) and -dP/dt(max) (cardiomyocytes, 126% +/- 29% of control; smooth muscle cells, 108% +/- 19% of control; skin fibroblasts, 99% +/- 16% control; P =.0001). CONCLUSIONS: Fetal cardiomyocytes transplanted into myocardial scar provided greater contractility and relaxation than fetal smooth muscle cells or fetal fibroblasts. The contractile and elastic properties of transplanted cells determine the degree of improvement in ventricular function achievable with cell transplantation.

The fate of a tissue-engineered cardiac graft in the right ventricular outflow tract of the rat.

OBJECTIVE: The synthetic materials currently available for the repair of cardiac defects are nonviable, do not grow as the child develops, and do not contract synchronously with the heart. We developed a beating patch by seeding fetal cardiomyocytes in a biodegradable scaffold in vitro. The seeded patches survived in the right ventricular outflow tract of adult rats. METHODS: Cultured fetal or adult rat heart cells (1 x 10(6) cells) were seeded into a gelatin sponge (15 x 15 x 1 mm), and the cell number was expanded in culture for 1 or 3 weeks, respectively. The free wall of the right ventricular outflow tract in syngeneic adult rats was resected and repaired with either unseeded patches or patches seeded with either fetal or adult cardiomyocytes (n = 10 for each group). The patches were examined histologically over a 12-week period. RESULTS: A significant inflammatory reaction was noted in the patch at 4 weeks as the scaffold dissolved. At 12 weeks, the gelatin scaffold had completely dissolved. Both types of the seeded cells were detected in the patch with 5-bromo-2'-deoxyuridine staining, and they maintained their continuity. Unseeded patches had an ingrowth of fibrous tissue. The patches became thinner between the fourth and the twelfth weeks in unseeded (P =.003), fetal (P =.0001), and adult (P =.07) cardiomyocyte groups as the scaffold dissolved. The control patch, but not the cell-seeded patches, was thinner than the normal right ventricular outflow tract. The endocardial surface area of each patch was covered with endothelial cells identified by factor VIII staining. CONCLUSIONS: A gelatin patch was used to replace the right ventricular outflow tract in syngeneic rats. The seeded cells survived in the right ventricular outflow tract after the scaffold dissolved 12 weeks after implantation. In addition, the unseeded patches encouraged the ingrowth of fibrous tissue as the scaffold dissolved and the patches remained completely endothelialized.

Optimal flow rates for retrograde warm cardioplegia.

Retrograde delivery of warm blood cardioplegia may improve nutrient cardioplegic flow beyond coronary obstructions, but may not adequately perfuse the right ventricle and the posterior left ventricle. To determine the optimal flow rate for warm retrograde cardioplegia, we assessed 62 patients undergoing elective coronary artery bypass in two studies. In the low flow study, administration of 50 ml/min (n = 9), 75 ml/min (n = 11), or 100 ml/min (n = 7) was associated with high lactate production and oxygen extraction during cardioplegic administration. At 50 minutes of cardioplegic arrest, the coronary venous effluent pH was low in all groups. In the high flow study, 30 patients all received flow rates of 100, 200, and 300 ml/min in randomized order during the crossclamp period. In addition, five patients received cardioplegia at a rate of 500 ml/min for the duration of the crossclamp period. Administration of 200 ml/min or higher minimized lactate production and maintained coronary venous pH within the physiologic range, but flows of 300 ml/min or higher did not increase oxygen use or reduce lactate or acid production. Patients in the low flow groups had significantly greater myocardial lactate release during cardioplegic infusion and after removal of the crossclamp than the high flow group. Warm retrograde cardioplegia should be delivered at flow rates of at least 200 ml/min during elective coronary artery bypass operations.

Angiogenesis by endothelial cell transplantation.

PURPOSE: Myocardial angiogenesis may improve regional perfusion and perhaps function after cardiac injury. We evaluated the effect of endothelial cell transplantation into a myocardial scar on angiogenesis and ventricular function, as an alternative to angiogenic gene or protein therapy. METHODS AND RESULTS: A transmural myocardial scar was created in the left ventricular free wall of rat hearts by cryoinjury. Allogeneic aortic endothelial cells were injected into the scar 2 weeks after cryoinjury. A cluster of transplanted cells was identified at the site of injection 1 day and 1 week after transplantation, but not after 2 weeks. The size of this cluster of transplanted cells decreased as vascular density in the transplanted scar tissue increased with time. Six weeks after transplantation, vascular density was significantly greater in transplanted hearts than in control hearts. Regional blood flow, by microsphere analysis, was greater in the transplanted rats. Systolic and diastolic ventricular function was similar between groups. In a second series of experiments, syngeneic aortic endothelial cells labeled with bromodeoxyuridine were transplanted 2 weeks after cryoinjury. Vascular density in the transplanted scar was greater than in controls. Labeled transplanted endothelial cells were identified forming part of the newly developed blood vessels. No difference in vascular density was found between allogeneic and syngeneic cell transplantation. Vascular endothelial growth factor was not expressed at greater levels in the transplanted cells or the myocardial scar. CONCLUSION: Transplanted endothelial cells stimulated angiogenesis, were incorporated into the new vessels, and increased regional perfusion in myocardial scar tissue, but did not improve global function in this cryoinjury rat model.