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.     

Transplantation of cryopreserved muscle cells in dilated cardiomyopathy: effects on left ventricular geometry and function

OBJECTIVE: Cell transplantation to prevent congestive heart failure in patients with inherited dilated cardiomyopathy might require the use of noncardiac donor cells unaffected by the genetic defect and cryopreservation to permit cell storage until the time of transplantation. However, the effects of cryopreservation on peripheral muscle cells harvested from a cardiomyopathic recipient and their subsequent ability to restore cardiac structure and function after transplantation are unknown. METHODS: Skeletal myoblasts and vascular smooth muscle cells from cardiomyopathic hamsters (delta-sarcoglycan-deficient BIO 53.58 hamster) and age-matched normal donor hamsters were isolated, expanded in culture, and cryopreserved. After reanimation in culture, cell morphology and growth rate were assessed and compared with values seen in noncryopreserved cells. A total of 4 x 10(6) previously cryopreserved skeletal myoblasts (n = 10) and vascular smooth muscle cells (n = 10) harvested from cardiomyopathic donors were then transplanted into the left ventricles of 17-week-old BIO 53.58 hamsters. Hearts injected with culture medium alone (n = 11) served as controls. Heart function was assessed 5 weeks after transplantation on a Langendorff apparatus, and left ventricular geometry was quantified by means of computerized planimetry. Staining with 5-bromo-2'-deoxyuridine identified the injected cells. RESULTS: Vascular smooth muscle cells from cardiomyopathic donors had an abnormal morphology and diminished growth rates in culture compared with vascular smooth muscle cells from normal donors. These markers of injury were exacerbated by cryopreservation. In contrast, vascular smooth muscle cells from normal donors and skeletal myoblasts from either cardiomyopathic or normal donors appeared normal in culture and were unaffected by cryopreservation. Both cryopreserved vascular smooth muscle cells and skeletal myoblasts from cardiomyopathic donors formed a viable muscle-resembling tissue that prevented wall thinning, limited left ventricular dilatation, and preserved global systolic function in hamsters with a genetic dilated cardiomyopathy. However, attenuation of cardiac remodeling and preservation of global heart function was greater after skeletal myoblast transplantation compared with vascular smooth muscle cell transplantation in parallel to the in vitro morphologic and growth characteristics of these cells. CONCLUSIONS: Cryostorage of healthy donor cells does not prevent the benefits of cell transplantation on limiting remodeling and preserving cardiac function in the failing heart. The health of donor cells in vitro predicts their subsequent benefits on cardiac structure and function after transplantation. Cryopreservation of donor cells might facilitate a clinically applicable and effective approach for ventricular restoration with cell-transplantation therapy for patients with inherited dilated cardiomyopathy.     

Normalization of coronary microvascular reactivity and improvement in myocardial perfusion by surgical vascular endothelial growth factor therapy combined with oral supplementation of l-arginine in a porcine model of endothelial dysfunction

OBJECTIVE: Vascular endothelial growth factor acts in part through nitric oxide release, the availability of which is decreased in endothelial dysfunction associated with advanced coronary artery disease. This could explain the relatively disappointing results of vascular endothelial growth factor therapy in clinical studies compared with animal studies. We examined the influence of L-arginine supplementation to vascular endothelial growth factor therapy on myocardial microvascular reactivity and perfusion in a porcine model of endothelial dysfunction. METHODS: Twenty-four pigs were fed either a normal (NORM, n = 8) or high-cholesterol diet with (CHOL-ARG, n = 8) or without (CHOL, n = 8) L-arginine. All pigs underwent ameroid placement on the circumflex artery and then 3 weeks later received surgical vascular endothelial growth factor treatment. Four weeks after treatment, endothelial-dependent coronary microvascular responses and lateral myocardial perfusion were assessed. Endothelial cell density was determined by means of immunohistochemistry. Vascular endothelial growth factor, endothelial nitric oxide synthase, and Akt levels were determined by means of immunoblotting. RESULTS: Pigs from the CHOL group showed endothelial dysfunction in the circumflex territory, which was normalized by L-arginine supplementation. Vascular endothelial growth factor treatment was ineffective in the CHOL group (circumflex/left anterior descending coronary artery blood flow ratios: 0.95 [rest] and 0.74 [pace] before-after treatment; P < .05 compared with the NORM group). Addition of L-arginine restored the angiogenic effect of vascular endothelial growth factor (ratios: 1.13 [rest] and 1.20 [pace]; P < .05) and was associated with increased endothelial cell density, as well as vascular endothelial growth factor, endothelial nitric oxide synthase, and Akt protein levels in the ischemic territory. CONCLUSIONS: L-Arginine supplementation can restore normal endothelium-dependent vasorelaxation and angiogenic response to vascular endothelial growth factor in a swine model of chronic myocardial ischemia with hypercholesterolemia-induced endothelial dysfunction. These findings suggest a putative role for L-arginine in combination with vascular endothelial growth factor therapy for end-stage coronary artery disease.     

Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy

OBJECTIVES: Cellular cardiomyoplasty with isolated skeletal myoblasts and bone marrow mononuclear cells is an encouraging therapeutic strategy for heart failure. We investigated the achievements accomplished with combined cell therapy of skeletal myoblast and bone marrow mononuclear cell transplantation to the ischemic canine myocardium. METHODS: Autologous skeletal myoblasts (1 x 10(8)) and autologous bone marrow mononuclear cells (3 x 10(6)) were injected directly into the damaged myocardium of canine hearts that had undergone 2 weeks of left anterior descending coronary artery ligation. Treatment groups were as follows: skeletal myoblasts plus bone marrow mononuclear cells (combined cell therapy, n = 4), myoblasts (n = 4), bone marrow mononuclear cells (n = 4), and medium only (n = 4). In similarly designed supporting experiments, angiogenic factor expression was evaluated by enzyme-linked immunosorbent assay after cell transplantation in rat hearts that had undergone left anterior descending coronary artery ligation. RESULTS: Four weeks after cell implantation, echocardiography demonstrated better cardiac performance with reduced left ventricular dilation and significantly improved ejection fraction in the combined cell therapy group compared with that seen in the other groups (pretreatment, 37.7% +/- 1.1%, vs combined cell therapy, 55.4% +/- 8.6%; myoblasts, 47.4% +/- 7.4%; bone marrow mononuclear cells, 44.4% +/- 6.7%; medium only [control], 34.4% +/- 5.4%; P < .05). A significantly high number of neovessels were observed in the group receiving combined cell therapy only (combined cell therapy, 45.5 +/- 12 x 10(2)/mm2; myoblasts, 26.5 +/- 8 x 10(2)/mm2; bone marrow mononuclear cells, 30.7 +/- 15 x 10(2)/mm2; medium only [control], 7.1 +/- 1 x 10(2)/mm2; P < .05). Immunostained sections expressed the skeletal specific marker myosin heavy chain, although they did not express the cardiac specific marker troponin T. Results of enzyme-linked immunosorbent assay showed the highest expression of vascular endothelial growth factor (combined cell therapy, 2.9 +/- 0.7 ng/g tissue; myoblasts, 0.24 +/- 0.7 ng/g tissue; bone marrow mononuclear cells, 1.9 +/- 0.2 ng/g tissue; medium only [control], 0.19 +/- 0.004 ng/g tissue; P < .05) and hepatocyte growth factor in the combined cell therapy hearts. CONCLUSIONS: Combined autologous cellular therapy induced both myogenesis and angiogenesis with enhancement of cardiac performance and reduction of cardiac remodeling, suggesting a capable strategy for treating severe ischemic cardiomyopathy clinically.     

Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ

BACKGROUND: Adult heart lacks stem cells and cannot effectively regenerate. In contrast, skeletal muscle is constantly undergoing repair. We proposed to transplant immature skeletal myoblasts into injured myocardium. METHODS: Approximately 7x10(6) soleus skeletal myoblasts were expanded in vitro from adult New Zealand White rabbits (n = 23) whose posterior left ventricle was cryoinjured to create a transmural lesion. Autologous myoblasts (n = 18) or saline (n = 5) was transplanted into the central cryolesion at the time of injury (n = 6) or 1 week later (n = 12). Hearts were harvested 2 weeks after injection. RESULTS: Myoblast transfer did not incur further morbidity. After cryolesion, grossly, a 1.6-cm epicardial hemorrhagic lesion could be seen. Histologically, the transmural lesion contained inflammatory cells and active scarring but no viable cardiomyocytes. Electron microscopy demonstrated a predominance of collagen and fibroblasts. Nine hearts contained multinucleated cells within the cryolesion that covered approximately 75% of the central cryolesion in 17% of animals. Immunohistochemical analysis confirmed their skeletal muscle origin. At the periphery of the lesion, isolated clusters of nonskeletal muscle cells could be visualized (n = 12) that resembled immature cardiocytes. CONCLUSIONS: Autologous skeletal myoblasts can regenerate viable striated tissue within damaged myocardium. Myoblast transfer warrants further investigation as a new method for improving myocardial performance within infarcted myocardium.     

Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion.

OBJECTIVE: The effect of transplanted mesenchymal stem cells (MSCs) on the left ventricular (LV) function and morphology in a rat myocardial infarct heart with reperfusion model were analyzed. METHODS: One week after 60 min of myocardial ischemia and reperfusion by left anterior descending artery (LAD) occlusion, 1.0x10(7) 6-diamidino-2-phenylindole (DAPI)-labeled MSCs were injected into the infarcted myocardium and compared with controls, and sham-operated rats, in which a cell-free serum medium was injected into the infarcted region or the myocardial wall, respectively. Measurement of vascular endothelial growth factor (VEGF) expression 1 week after MSC injection using Western blot analysis (n=5), and immunohistochemical staining using HE staining and fluorescent microscopy of the DAPI-positive regions from MSC implantation, cTnT immunostaining of potential myocardial-like cells, and SM-actin and CD31 immunostaining demonstrating neovascular transformation of implanted MSCs 1 week, 2 weeks and 4 weeks after transplantation (n=5). Hemodynamic measurements were performed after 4 weeks in vivo. Subsequently, hearts were quickly removed and cut for histological analysis using HE staining with measurement of the infarcted LV-area, the LV-wall thickness within the scar segment compared to non-infarcted scar segments, and the capillary density counting capillary vessels with 400x light microscopy (n=10). RESULTS: Measurement of hemodynamics 4 weeks after transplantation in vivo showed LV function to be significantly greater in MSCs than in the control group. Semi-quantitative histomorphometric examinations showed a significantly lower infract size, a greater LV-wall thickness, and a lower Hochman-Choo expansion index in the MSC-treated group compared to the control group. Immunofluorescence demonstrated that transplanted MSCs were positive for cTnT, suggesting that a small number of transplanted MSCs can differentiate into cardiomyocytes. Other MSCs were positive for CD31 and SM-actin. The transplanted MSCs in MI area had significantly higher expression rates of cTnT, CD31 and SM-actin 2 weeks after transplantation. HE staining showed marked augmentation of neovascularization in the MSC group. Semi-quantitative analysis demonstrated that capillary density was significantly higher in the MSC group than in the control group. CONCLUSION: Implanted MSCs could improve cardiac structure and function through the combined effect of myogenesis and angiogenesis.     

Enhanced cytoprotection and angiogenesis by bone marrow cell transplantation may contribute to improved ischemic myocardial function.

OBJECTIVES: Heat shock proteins (HSPs) are cytoprotective proteins. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor. This study aimed to elucidate the possible role of cytoprotection and angiogenesis on cardiac function after bone marrow cell transplantation (BMT). METHODS: Myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation. A total of 5 x 10(6) bone marrow-mononuclear cells were transplanted into the ischemic zone by direct injection. At 1, 3, 7, 14 and 28 days post-transplantation, cardiac function was evaluated by echocardiography. The expressions of HSP32, HSP70 and VEGF were assessed by immunofluorescence and RT-PCR. The number of vessels was examined by immunohistochemistry. The differentiation of the transplanted cells was determined by immunofluorescence. RESULTS: Echocardiography showed BMT led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction and fraction of shortening. Immunofluorescence revealed that the expressions of HSP32, HSP70 and VEGF were promoted in both transplanted bone marrow cells and recipient cardiomyocytes. RT-PCR showed that the mRNA expression levels of HSP32, HSP70 and VEGF in the BMT group were markedly higher in comparison with injection of peripheral blood cells or saline (P<0.01) by day 7. Seven days later, the vessel count showed that angiogenesis had been induced to a significantly greater degree in the BMT groups. Fourteen days later, specific markers for myocardial or vascular endothelial cells were detected in the transplanted bone marrow cells. CONCLUSIONS: BMT upregulated the expressions of HSP32, HSP70 and VEGF in both transplanted bone marrow cells and recipient endogenous cardiomyocytes in the early phase post-transplantation. This enhanced cytoprotection and angiogenesis, and contributed to the functional recovery following cardiac infarction. In the late phase, the transplanted bone marrow cells might differentiate into both myocardial and vascular endothelial cells that enhanced the ischemic cardiac function further.     

VEGF121体外转染大鼠骨骼肌成肌细胞的实验研究

目的研究血管内皮生长因子基因（VEGF121）在原代培养大鼠骨骼肌成肌细胞中的转染表达。方法通过脂质体介导将VEGF121基因转染到原代培养的大鼠骨骼肌成肌细胞中。经RT-PCR、Weaterrv Blot及免疫荧光检测转基因成肌细胞的基因表达情况。结果成肌细胞在基因转染后24h即出现VEGF121的表达，于48～72h达表达高峰，以后逐渐下调，维持表达2周左右。转染后的成肌细胞表达VEGF121蛋白质产物明显增加。结论经脂质体介导转染VEGF121基因的大鼠骨骼肌成肌细胞在体外有良好的因基表达，为VEGF基因修饰成肌细胞移植奠定基础。     

Autologous heart cell transplantation improves cardiac function after myocardial injury

Background. Fetal ventricular cardiomyocyte transplantation into a cardiac scar improved ventricular function, but these cells were eventually eliminated by rejection. We therefore examined the feasibility of autologous adult heart cell transplantation.

Methods. A transmural scar was produced in the left ventricular free wall of adult rats by cryoinjury. The left atrial appendage was harvested, and the atrial heart cells were cultured and their number expanded ex vivo. Three weeks after cryoinjury, either a cell suspension (2 × 10⁶ cells, n = 12 rats, transplant group) or culture medium (n = 10 rats, control group) was injected into the scar. Rats having a sham operation (n = 5) did not undergo cryoinjury or transplantation with cells or culture medium.

Results. Five weeks after injection, ventricular function was evaluated in a Langendorff preparation, measuring systolic, diastolic, and developed pressures over a range of intraventricular balloon volumes. Systolic and developed pressures were greater in the transplant group than in the control group (p = 0.0001). Rats with a sham operation had the greatest systolic, diastolic, and developed pressures (p = 0.0001). Histologic studies demonstrated survival of the transplanted heart cells within the scar. The area of the scar was smaller (p = 0.0003) and its thickness greater (p = 0.0003) in rats in the transplant group. Left ventricular chamber volume was smaller in the transplant group (p = 0.043).

Conclusions. Transplantation of autologous cultured adult atrial heart cells limited scar thinning and dilatation and improved myocardial function compared with results in control hearts. This technique may lead to a novel therapy to prevent scar expansion after a myocardial infarction and prevent the development of congestive heart failure.     

Systolic contraction within aneurysmal rabbit myocardium following transplantation of autologous skeletal myoblasts

OBJECTIVE: Transplantation of autologous skeletal myoblasts (SKMB) into infarcted heart (or cellular cardiomyoplasty, CCM) augments myocardial performance in animal models of myocardial infarction. However, the effect of CCM in the setting of ventricular aneurysm has not been evaluated. This study analyzes the effects of transplanted SKMB on regional wall motion in a rabbit model of postinfarct ventricular aneurysm. We hypothesize that CCM, performed early after myocardial infarction, prevents the progression of dyskinetic wall motion. METHODS: Twenty-six rabbits underwent apical left ventricular cryoinfarction and soleus muscle biopsy for in vitro isolation of skeletal myoblasts. At 2 weeks postinfarct, the presence of ventricular aneurysm was detected in 23/26 animals by sonomicrometry and micromanometry. Seventeen of 23 animals were randomized to receive either 108 autologous myoblasts (CCM) or vehicle. Regional stroke work, percent systolic shortening, and synchronicity of regional wall motion were determined prior to, and four weeks following, injection (CCM; n = 9; vehicle, n = 8). Wall motion was evaluated under baseline and stress (dobutamine, 10 (g/kg/min) conditions. Six animals did not undergo randomization, but their hearts were used to measure the size of infarction. RESULTS: Four weeks following treatment of animals with ventricular aneurysm, systolic contractile activity was present in most animals treated with myoblasts but in none treated with vehicle (5/7 versus 0/6, respectively, P < 0.05). Dobutamine tended to accentuate the differences seen at baseline between the groups. CONCLUSIONS: This study demonstrates a high incidence of systolic contractile activity in a previously aneurysmal region of myocardium following CCM and may represent a novel therapy for the prevention and treatment of postinfarct aneurysm.     

Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia

OBJECTIVE: We aimed to achieve angiogenic synergism between human vascular endothelial growth factor 165 (VEGF 165 ) and angiopoietin-1 (Ang-1) using a new adenoviral bicistronic vector concurrently with cell therapy to repair an ischemically damaged hind limb in a rabbit model. METHODS: Rabbit autologous primary skeletal myoblasts were isolated and labeled with retrovirally transduced LacZreporter gene, 4,6-diamidino-2-phenylindole (DAPI), and 5-bromo-2'-deoxyuridine (BrdU). Hind limb ischemia was created in 48 female New Zealand White rabbits by means of femoral artery ligation at 8 different places, and was assessed at angiography. Animals were randomized to receive intramuscular injection of either Dulbeco's Modified Eagle Medium (DMEM;group 1, n = 8), nontransduced myoblasts (group 2, n = 10), or myoblasts transduced with Ad-Null (group 3, n = 10), Ad-VEGF (group 4, n = 10), or Ad-Bicis (group 5, n = 8). Six weeks after treatment neovascularization in the limb was assessed at angiography. The animals were euthanized, and tissue was harvested for histologic study. RESULTS: Extensive transplanted myoblast survival was observed in all cell-transplanted groups, as visualized with DAPI, BrdU, and LacZ staining. Angiographic blood vessel count revealed enhanced neovascularization in group 5 (25.14 +/- 5.14) compared with group 4 (13.62 +/- 4.52), group 3 (6.09 +/- 0.09), group 2 (4.67 +/- 3.49), and group 1 (3.18 +/- 7.76). Immunostaining for von Willebrand factor confirmed significantly increased capillary density ( P < .01) at high-power microscopic field in group 5 (19.04 +/- 1.59) compared with group 4 (15.31 +/- 1.55), group 3 (6.53 +/- 0.97), group 2 (5.69 +/- 0.51), and group 1 (3.03 +/- 0.20). CONCLUSION: Simultaneous expression of VEGF and Ang-1 from bicistronic vector transduced skeletal myoblasts potently stimulated enhanced functional neovascularization in a rabbit model of limb ischemia.     

Cell transplantation to prevent heart failure: a comparison of cell types

BACKGROUND: Autologous cell transplantation may restore viable muscle after a myocardial infarction. We compared the effect of three cell types or an angiotensin-converting enzyme (ACE) inhibitor on preservation of ventricular function after cardiac injury. METHODS: A uniform transmural myocardial scar was created in adult rats by cryoinjury. Three weeks later the rats were randomly assigned to one of four blinded treatments: transplantation with 5 x 10(6) aortic smooth muscle cells (SMC, n = 12), ventricular heart cells (VHC, n = 13), skeletal muscle cells (SKC, n = 13) or culture medium alone (control, n = 11). The ACE inhibitor group (n = 8) received enalapril (1.0 mg/kg per day), also beginning 3 weeks after cryoinjury. Five and 12 weeks after transplantation, left ventricle (LV) function was assessed in a Langendorff apparatus, and histologic and immunohistological evaluation of the LV scars was performed. RESULTS: At 5 weeks, greater scar elastin content and better LV function was noted with cell transplantation or ACE inhibitor therapy compared with control rats (p < 0.05). Twelve weeks after transplantation, cell-transplanted rats still had greater elastin content and better LV function than control rats, although elastin content and LV function had declined in ACE inhibitor-treated animals to levels below those observed in control rats (p < 0.05). CONCLUSIONS: Transplantation of SMC, VHC, and SKC preserved ventricular function equivalent to the effects of an ACE inhibitor. Muscle cell transplantation, but not ACE inhibitor therapy, continues to be effective later after cryoinjury. No differences were detected between the muscle cells.     

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.     

Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function

OBJECTIVES: Transplantation of fetal cardiomyocytes improves function of infarcted myocardium but raises availability, immunologic, and ethical issues that justify the investigation of alternate cell types, among which skeletal myoblasts are attractive candidates. METHODS: Myocardial infarction was created in rats by means of coronary artery ligation. One week later, the animals were reoperated on and intramyocardially injected with culture growth medium alone (controls, n = 15), fetal cardiomyocytes (5 x 10(6) cells, n = 11), or neonatal skeletal myoblasts (5 x 10(6) cells, n = 16). The injections consisted of a 150-microL volume and were made in the core of the infarct, and the animals were immunosuppressed. Left ventricular function was assessed by echocardiography immediately before transplantation and 1 month thereafter. Myoblast-transplanted hearts were then immunohistologically processed for the expression of skeletal muscle-specific embryonic myosin heavy chain and cardiac-specific connexin 43. RESULTS: The left ventricular ejection fraction markedly increased in the fetal and myoblast groups from 39.3% +/- 3.9% to 45% +/- 3.4% (P =.086) and from 40.4% +/- 3.6% to 47.3% +/- 4.4% (P =.034), respectively, whereas it decreased in untreated animals from 40.6% +/- 4% to 36.7% +/- 2.7%. Transplanted myoblasts could be identified in all animals by the positive staining for skeletal muscle myosin. Conversely, clusters of connexin 43 were not observed on these skeletal muscle cells. CONCLUSIONS: These results support the hypothesis that skeletal myoblasts are as effective as fetal cardiomyocytes for improving postinfarction left ventricular function. The clinical relevance of these findings is based on the possibility for skeletal myoblasts to be harvested from the patient himself.     

Amelioration of microvascular myocardial ischemia by gene transfer of vascular endothelial growth factor in rabbits

OBJECTIVES: Restoration of coronary blood flow by angiogenesis may offer a new approach to intractable ischemic heart disease. In the present study, we investigated the angiogenic effects of gene transfer of vascular endothelial growth factor 165 on microvascular myocardial ischemia. METHODS: A rabbit model of microvascular myocardial ischemia was created by plugging coronary microvessels with microspheres (15 microm in diameter, 2.8 x 10(5)/kg, n = 29). Gene transfer was performed by semi-selective intracoronary injection of recombinant adenovirus expressing vascular endothelial growth factor 165 forty minutes after microsphere injection (n = 9). RESULTS: Microsphere injection reduced myocardial perfusion (78% +/- 9% of baseline tissue flow) and diminished myocardial contraction (61% +/- 12% of the baseline ejection fraction) and cardiac performance (elevated left ventricular end-diastolic pressure and decreased systemic flow) in the acute phase. At 17 +/- 3 days, gene transfer of vascular endothelial growth factor 165 had had the following effects: (1) promoted coronary angiogenesis as evidenced by myocardial flow above the baseline (121% +/- 24%), (2) increased vascular density revealed by synchrotron radiation microangiography and histologic analysis, (3) ameliorated the degree of myocardial ischemia as evidenced by myocardial lactate content and the extent of histologic necrosis, and (4) restored heart function as evidenced by increased ejection fraction (95% +/- 10%), reduced left ventricular end-diastolic pressure, and restored body weight. CONCLUSIONS: In vivo vascular endothelial growth factor 165 gene transfer promoted angiogenesis and was an effective approach to treating microvascular myocardial ischemia.     

Enhancement of the functional benefits of skeletal myoblast transplantation by means of coadministration of hypoxia-inducible factor 1alpha

OBJECTIVE: Early cell death remains a major limitation of skeletal myoblast transplantation. Because the poor vascularization of the target scars contributes to cell loss, we assessed the effects of combining skeletal myoblast transplantation with administration of hypoxia-inducible factor 1alpha, a master gene that controls the expression of a wide array of angiogenic factors. METHODS: A myocardial infarction was created in 56 rats by means of coronary artery ligation. Eight days later, rats were randomly allocated to receive in-scar injections of culture medium (control animals, n = 11), skeletal myoblasts (5 x 10(6) , n = 13), adenovirus-encoded hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL, n = 7), or skeletal myoblasts (5 x 10(6)) in combination with an empty vector (n = 3) or active hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL, n = 13). A fifth group (n = 9) underwent a staged approach in which hypoxia-inducible factor 1alpha (1.0 x 10(10) pfu/mL) was injected at the time of infarction, followed 8 days later by skeletal myoblasts (5 x 10(6)). Left ventricular function was assessed echocardiographically before transplantation and 1 month thereafter. Explanted hearts were then processed for the immunohistochemical detection of myotubes, quantification of angiogenesis, myoblast engraftment, and cell survival. RESULTS: Baseline ejection fractions were not significantly different among groups (35%-40%). One month later, ejection fraction had decreased from baseline in control hearts and in those injected with hypoxia-inducible factor 1alpha. In contrast, it did not deteriorate after injections of skeletal myoblasts alone or combined with either the empty vector or active hypoxia-inducible factor 1alpha administered sequentially. The most striking change occurred in the skeletal myoblast plus hypoxia-inducible factor 1alpha combined group in which ejection fraction increased dramatically (by 27%) above baseline levels and was thus markedly higher than in all other groups ( P = .0001 and P = .001 vs control animals and animals receiving hypoxia-inducible factor 1alpha, respectively). Compared with skeletal myoblasts alone, the coadministration of hypoxia-inducible factor 1alpha resulted in a significantly greater degree of angiogenesis, cell engraftment, and cell survival. CONCLUSION: Induction of angiogenesis is an effective means of potentiating the functional benefits of myoblast transplantation, and hypoxia-inducible factor 1alpha can successfully achieve this goal.     

VEGF expression in osteosarcoma correlates with vascular permeability by dynamic MRI

Dynamic enhanced magnetic resonance imaging has been used to assess tumor angiogenesis in osteosarcoma. Vascular endothelial growth factor has been shown to correlate with pulmonary metastasis and a poor prognosis in osteosarcoma. The purpose of this investigation was to determine whether vascular endothelial growth factor expression in osteosarcoma correlates with vascular permeability detected by dynamic enhanced magnetic resonance imaging and to explore the role of dynamic enhanced magnetic resonance imaging as a noninvasive means of assessing tumor angiogenic activity. Fifty-five osteosarcoma patients with osteosarcoma enrolled in a treatment protocol that included dynamic enhanced magnetic resonance imaging. In 15 patients, tumor tissues were available for vascular endothelial growth factor immunohistochemical studies. A two-compartment model used the exchange rate constants (kep) between the plasma and tumor compartments to quantify vascular permeability during dynamic magnetic resonance imaging studies. Immunohistochemical staining for vascular endothelial growth factor was graded according to the intensity and number of positively stained cells. Vascular endothelial growth factor-positive tumors showed higher mean vascular permeability when compared with vascular endothelial growth factor-negative tumors. Vascular permeability also correlated with increasing vascular endothelial growth factor expression. The preliminary results in this study show an association between vascular endothelial growth factor and dynamic MR signal enhancement in osteosarcoma. Dynamic enhanced magnetic resonance imaging should be investigated as a means to prognosticate osteosarcoma patients with osteosarcoma according to their tumor angiogenic activity.     

Vascular endothelial growth factor and basic fibroblast growth factor in children with cyanotic congenital heart disease

OBJECTIVE: Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor. METHODS: Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay. RESULTS: Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02). CONCLUSION: Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.