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.

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.

Lactate release during reperfusion predicts low cardiac output syndrome after coronary bypass surgery.

BACKGROUND: Cardioplegic arrest induces anaerobic myocardial metabolism with a net production of lactate from glycolysis. However, persistent lactate release during reperfusion suggests a delayed recovery of normal aerobic metabolism and may lead to depressed myocardial function necessitating inotropic or intraaortic balloon pump support (low output syndrome [LOS]). We examined the relation between perioperative myocardial metabolism and postoperative clinical outcomes in patients undergoing isolated coronary artery bypass surgery (CABG). METHODS: We reviewed 623 patients who were enrolled in clinical studies evaluating perioperative myocardial metabolism between 1983 and 1996. Arterial and coronary sinus blood samples were obtained intraoperatively to assess myocardial metabolism. Clinical data regarding patient demographics and postoperative outcomes were prospectively collected and entered into our institutional database. RESULTS: Low output syndrome developed in 36 patients (5.8%). Myocardial lactate release was higher in these patients compared with those who did not develop postoperative LOS. Advanced age and poor preoperative left ventricular function were independent predictors of lactate release during reperfusion. Persistent lactate release after 5 minutes of reperfusion was the only independent predictor of postoperative LOS in this low-risk population. CONCLUSIONS: Persistent lactate release during reperfusion occurs in a significant proportion of low-risk patients undergoing isolated CABG and is an independent predictor of postoperative low cardiac output syndrome. Persistent lactate release during reperfusion suggests a delayed recovery of aerobic myocardial metabolism and may be related to intraoperative misadventure or inadequate myocardial protection. Myocardial lactate release may be useful as an alternative end-point in clinical trials evaluating perioperative myocardial protection.

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.

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.

Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury.

BACKGROUND: This study was designed to determine the optimal time for cell transplantation after myocardial injury. METHODS: The left ventricular free wall of adult rat hearts was cryoinjured and the animals were sacrificed at 0, 1, 2, 4, and 8 weeks for histologic studies. Fetal rat cardiomyocytes (transplant) or culture medium (control) were transplanted immediately (n = 8), 2 weeks (n = 8), and 4 weeks (n = 12) after cryoinjury. At 8 weeks, rat heart function, planimetry, and histologic studies were performed. RESULTS: Cryoinjury produced a transmural injury. The inflammatory reaction was greatest during the first week but subsided during the second week after cryoinjury. Scar size expanded (p < 0.01) at 4 and 8 weeks. Cardiomyocytes transplanted immediately after cryoinjury were not found 8 weeks after cryoinjury. Scar size and myocardial function were similar to the control hearts. Cardiomyocytes transplanted at 2 and 4 weeks formed cardiac tissue within the scar, limited (p < 0.01) scar expansion, and had better (p < 0.001) heart function than the control groups. Developed pressure was greater (p < 0.01) in the hearts with transplanted cells at 2 weeks than at 4 weeks. CONCLUSIONS: Cardiomyocyte transplantation was most successful after the inflammatory reaction resolved but before scar expansion.

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.

Autologous smooth muscle cell transplantation improved heart function in dilated cardiomyopathy.

BACKGROUND: Transplantation of myocytes into scarred myocardium has been shown to inhibit ventricular remodeling and maintain myocardial contractility. However, the effect of cell transplantation on hearts with global rather than regional dysfunction is unknown. Therefore, we evaluated the effect of transplantation of autologous smooth muscle cells on the morphometry and function of dilated cardiomyopathic hearts. METHODS: Smooth muscle cells were isolated from the ductus deferens of 13-week-old BIO 53.58 hamsters with dilated cardiomyopathy, and cultured for 4 weeks before transplantation. Smooth muscle cells (4 x 10(6) cells) or culture medium were injected into 17-week-old animals in the transplantation and control groups (n = 12 each), respectively. Prelabeling of the smooth muscle cells with 5-bromo-2'-deoxyuridine was performed before transplantation in a group of transplanted hamsters. Another group (sham, n = 12) underwent the operation but did not receive an injection either of smooth muscle cells or of culture medium. Four weeks after transplantation, heart function was evaluated in a Langendorff preparation. RESULTS: Musclelike tissue, labeled with 5-bromo-2'-deoxyuridine, was found at the site of transplantation in the cell-transplanted animals. The cell-transplanted hearts were smaller (p < 0.001), and had greater developed pressures and maximum rate of increase of left ventricular pressure (both p < 0.001) than control and sham hearts. Control hamsters injected with culture medium did not differ from sham-operated animals. CONCLUSIONS: Transplantation of autologous smooth muscle cells prevented cardiac dilatation and improved ventricular function in hamsters with dilated cardiomyopathy.