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1.
Am J Physiol Heart Circ Physiol ; 289(5): H2089-96, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16219813

ABSTRACT

We evaluated the impact of donor age on the efficacy of myocardial cellular therapy for ischemic cardiomyopathy. Characteristics of smooth muscle cells (SMC), bone marrow stromal cells (MSCs), and skeletal muscle cells (SKMCs) from young, adult, and old rats were compared in vitro. Three weeks after coronary ligation, 3.5 million SMCs (n = 11) or MSCs (n = 9) from old syngenic rats or culture medium (n = 6) were injected into the ischemic region. Five weeks after implantation, cardiac function was assessed by echocardiography and the Langendorff apparatus. In the in vitro study, the numbers and proliferation of MSCs from fresh bone marrow and SKMCs from fresh tissue but not SMCs were markedly diminished in old animals (P < 0.05 both groups). SKMCs from old animals did not reach confluence. After treatment with 5-azacytidine (azacitidine), the myogenic potential of old MSCs was decreased compared with young MSCs. In the in vivo study, both SMC and MSC transplantation induced significant angiogenesis compared with media injections (P < 0.05 both groups). Transplantation of SMCs but not MSCs prevented scar thinning (P = 0.03) and improved ejection fraction and fractional shortening (P < 0.05). Load-independent indices of cardiac function in a Langendorff preparation confirmed improved function in the aged SMC group (P = 0.01) but not in the MSC group compared with the control group. In conclusion, donor age adversely impacts the efficacy of cellular therapy for myocardial regeneration and is cell-type dependent. SMCs from old donors retain their ability to improve cardiac function after implantation into ischemic myocardium.


Subject(s)
Aging/physiology , Bone Marrow Transplantation/physiology , Cell- and Tissue-Based Therapy , Muscle, Smooth, Vascular/physiology , Myocytes, Cardiac/transplantation , Animals , Cell Separation , Cells, Cultured , Colony-Forming Units Assay , Coronary Vessels/physiology , Heart Failure/therapy , Ligation , Male , Muscle Fibers, Skeletal/transplantation , Myocardium/cytology , Rats , Rats, Inbred Lew , Ventricular Function, Left/physiology
2.
Ann Thorac Surg ; 79(6): 2056-63, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15919309

ABSTRACT

BACKGROUND: We have previously reported that transplantation of vascular endothelial growth factor transfected cells into myocardial scar enhances angiogenesis. We evaluated the effect of transplanted cell type, time, and region of the heart on expression of the vascular endothelial growth factor receptors fms-like tyrosine kinase-1 (flt-1) and fetal liver kinase-1 (flk-1). METHODS: Lewis rats underwent myocardial cryoinjury 3 weeks before transplantation with heart cells (a mixed culture of cardiomyocytes, smooth muscle cells, endothelial cells, and fibroblasts), vascular endothelial growth factor transfected heart cells, skeletal myoblasts, vascular endothelial growth factor transfected skeletal myoblasts, or medium (controls) (N = 13 each). Flt-1 and flk-1 expression in the scar, border zone, and normal myocardium were evaluated at 3 days and 1, 2, and 4 weeks by quantitative polymerase chain reaction. Transplanted cells, vascular endothelial growth factor, flt-1, and flk-1 were identified by immunohistology. RESULTS: Flt-1 and flk-1 levels were low in all areas of control hearts. Upregulation of flt-1 and flk-1 after cell transplantation occurred primarily in host cells in the border zone rather than the scar (zone, p < 0.0001). Flt-1 and flk-1 expression was doubled by heart cells and skeletal myoblasts and increased eightfold by vascular endothelial growth factor transfected heart cells and skeletal myoblasts (group, p < 0.0001). Flk-1 expression peaked at 1 week, whereas flt-1 peaked at 2 weeks (time, p < 0.0001). CONCLUSIONS: Flk-1 and flt-1 upregulation may mediate the angiogenic effect of cell transplantation and are augmented by vascular endothelial growth factor transgene expression, perhaps through a paracrine effect. Optimizing the angiogenic response to cell transplantation may maximize the benefit of cell transplantation strategies.


Subject(s)
Cell Transplantation , Gene Expression Regulation , Genetic Therapy , Myocardium/pathology , Neovascularization, Physiologic/genetics , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Animals , Disease Models, Animal , Endothelial Cells/transplantation , Female , Fibroblasts/transplantation , Heart Diseases/therapy , Male , Myocytes, Cardiac/transplantation , Paracrine Communication , Rats , Rats, Inbred Lew , Transfection , Transgenes , Up-Regulation , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics
3.
J Thorac Cardiovasc Surg ; 129(4): 904-11, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15821662

ABSTRACT

BACKGROUND: Cell transplantation improves heart function after myocardial infarction. This study investigated the survival of implanted cells in normal and infarcted myocardium. METHODS: Male rat aortic smooth muscle cells were cultured. For the in vitro study, male smooth muscle cells mixed with female smooth muscle cells or male smooth muscle cells injected into a piece of female rat myocardium were used to evaluate the accuracy of quantitative real-time polymerase chain reaction to measure Y chromosomes. For the in vivo study, 2 million live or dead male smooth muscle cells were injected into normal or infarcted female myocardium. At 1 hour and 1 and 4 weeks after transplantation, hearts, lungs, and kidneys were harvested for measurement of Y chromosomes. RESULTS: In vitro, the accuracy of polymerase chain reaction measurement was excellent in cultured cells (r2 = 0.996) and the myocardium (r2 = 0.786). In vivo, 1 hour after 2 x 10(6) cell implantation, live cell numbers decreased to 1.0 +/- 0.2 x 10 6 and 1.1 +/- 0.3 x 10(6) , and dead cell numbers decreased to 0.9 +/- 0.2 x 10(6) and 0.8 +/- 0.2 x 10(6) in the normal and infarcted myocardium, respectively (P < .01 for all groups). Lungs and kidneys contained 8.5% and 1.5% of the implanted cells, but no cells were detected at 1 week. At 1 week, no dead smooth muscle cells were detected in the normal or infarcted myocardium. The numbers of live cells at 1 and 4 weeks were 0.48 +/- 0.06 x 10(6) and 0.27 +/- 0.07 x 10(6) in normal myocardium and 0.29 +/- 0.08 x 10(6) and 0.18 +/- 0.05 x 10(6) in infarcted myocardium. CONCLUSIONS: One hour after implantation, only 50% of smooth muscle cells remained in the implanted area. Some implanted cells deposited in other tissue. Implanted cell survival progressively decreased during the 4-week study.


Subject(s)
Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/transplantation , Animals , Antimetabolites , Brain/pathology , Bromodeoxyuridine , Cell Death , Cell Survival , Cells, Cultured , DNA/genetics , Female , Graft Survival , Kidney/pathology , Lung/pathology , Male , Myocardial Infarction/surgery , Myocardium/pathology , Polymerase Chain Reaction , Rats , Rats, Inbred Lew , Time Factors , Y Chromosome/genetics
4.
Am J Physiol Heart Circ Physiol ; 288(6): H2819-27, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15681698

ABSTRACT

Ventricular dilation after myocardial infarction can cause heart failure. Increasing strength and elasticity in the infarct region might prevent ventricular dilation. Because elastin provides strength, extensibility, and resilience to tissues and maintains tissue architecture, we studied the effect of elastin expression in the infarct on scar expansion and heart function. COS-7 cells transfected with a plasmid with an elastin gene fragment or a vector were seeded into a Gelfoam mesh and cultured. Mechanical stretch test (n = 5/group) showed that the elastin mesh was more elastic (P < 0.05) and tensile (P < 0.05) than the vector mesh. In an in vivo study in rats, 6 days after left anterior descending coronary artery ligation, COS-7 cells (Cell group, n = 7) or COS-7 cells with elastin gene (Elastin group, n = 9) or vector (Vector group, n = 9) were transplanted into the infarct; infarcted rats served as controls (n = 7). Over 8 wk the Cell group did not demonstrate effects on scar expansion and deterioration of heart function vs. controls. In contrast, infarct expansion was smaller and heart function was better maintained in the Elastin group vs. the Vector group (P < 0.05). At 8 wk after cell transplantation Langendorff data showed that the Elastin group had greater (P < 0.01) developed pressure and a smaller left ventricular volume than the Vector group. Western blot and histology showed accumulated elastin in the Elastin group infarct. Changing the extracellular matrix composition of a myocardial infarct by increasing elastin fragment content attenuated scar expansion, ventricular dilation, and onset of heart dysfunction.


Subject(s)
Cicatrix/prevention & control , Elastin/genetics , Heart/physiology , Myocardial Infarction/metabolism , Animals , Aorta , Base Sequence , COS Cells , Chlorocebus aethiops , DNA Primers , Extracellular Matrix/metabolism , Genetic Vectors , Heart/physiopathology , Peptide Fragments/genetics , Rats , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection
5.
Am J Physiol Heart Circ Physiol ; 287(6): H2840-9, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15331370

ABSTRACT

The functional benefit of cell transplantation after a myocardial infarction is diminished by early cell losses. IGF-1 enhances cell proliferation and survival. We hypothesized that IGF-1-transfected smooth muscle cells (SMCs) would enhance cell survival and improve engraftment after cell transplantation. The IGF-1 gene was transfected into male SMCs and compared with SMCs transfected with a plasmid vector (vector control) and nontransfected SMCs (cell control). IGF-1 mRNA (n=10/group) and protein levels (n=6/group) were higher (P <0.05 for all groups) at 3, 7, and 14 days compared with controls. VEGF was also increased in parallel to enhanced IGF-1 expression. IGF-1-transfected cells demonstrated greater cell proliferation, stimulated angiogenesis, and decreased caspase-3 activity after simulated ischemia and reperfusion (P <0.05 for all groups compared with vector or cell controls). A uniform left ventricular injury was produced in female rats using a cryoprobe. Three weeks later, 2 x 10(6) cells from three groups were implanted into the scar. One week later, IGF-1-transfected SMCs had increased myocardial IGF-1 and VEGF levels, increased Bcl2 expression, limited cell apoptosis, and enhanced vessel formation in the myocardial scar compared with the two control groups (P <0.05 for all groups). The proportion of SMCs surviving in the implanted region was greater (P <0.05) in the IGF-1-transfected group than in the vector or cell controls. Gene enhancement with IGF-1 improved donor cell proliferation, survival, and engraftment after cell transplantation, perhaps mediated by enhanced angiogenesis and reduced apoptosis.


Subject(s)
Insulin-Like Growth Factor I/genetics , Myocardial Infarction/therapy , Myocytes, Smooth Muscle/physiology , Myocytes, Smooth Muscle/transplantation , Animals , Aorta/cytology , Apoptosis/physiology , Cell Division/physiology , Cell Survival/physiology , Female , Gene Expression , In Vitro Techniques , Male , Myocardium/cytology , Myocytes, Smooth Muscle/cytology , Rats , Rats, Inbred Lew , Transfection , Vascular Endothelial Growth Factor A/genetics
6.
Circulation ; 110(16): 2401-9, 2004 Oct 19.
Article in English | MEDLINE | ID: mdl-15262835

ABSTRACT

BACKGROUND: Despite the mounting clinical burden of heart failure, the biomolecules that control myocardial tissue remodeling are poorly understood. TIMP-3 is an endogenous inhibitor of matrix metalloproteinases (MMPs) that has been found to be deficient in failing human myocardium. We hypothesized that TIMP-3 expression prevents maladaptive tissue remodeling in the heart, and accordingly, its deficiency in mice would alone be sufficient to trigger progressive cardiac remodeling and dysfunction similar to human heart failure. METHODS AND RESULTS: Mice with a targeted timp-3 deficiency were evaluated with aging and compared with age-matched wild-type littermates. Loss of timp-3 function triggered spontaneous LV dilatation, cardiomyocyte hypertrophy, and contractile dysfunction at 21 months of age consistent with human dilated cardiomyopathy. Its absence also resulted in interstitial matrix disruption with elevated MMP-9 activity, and activation of the proinflammatory tumor necrosis factor-alpha cytokine system, molecular hallmarks of human myocardial remodeling. CONCLUSIONS: TIMP-3 deficiency disrupts matrix homeostasis and the balance of inflammatory mediators, eliciting the transition to cardiac dilation and dysfunction. Therapeutic restoration of myocardial TIMP-3 may provide a novel approach to limit cardiac remodeling and the progression to failure in patients with dilated cardiomyopathy.


Subject(s)
Cardiomyopathy, Dilated/enzymology , Tissue Inhibitor of Metalloproteinase-3/deficiency , ADAM Proteins , ADAM17 Protein , Animals , Cardiomyopathy, Dilated/diagnostic imaging , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , Collagen/analysis , Disease Progression , Extracellular Matrix/chemistry , Homeostasis , Hypertrophy , Macrophages/pathology , Matrix Metalloproteinase 9/analysis , Metalloendopeptidases/analysis , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Cardiovascular , Myocardial Contraction , Myocytes, Cardiac/pathology , Nitrites/analysis , Receptors, Tumor Necrosis Factor, Type II/analysis , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/physiology , Tumor Necrosis Factor-alpha/analysis , Ultrasonography
7.
Cell Transplant ; 13(2): 169-77, 2004.
Article in English | MEDLINE | ID: mdl-15129763

ABSTRACT

Autologous smooth muscle cell (SMC)-seeded biodegradable scaffolds could be a suitable material to repair some pediatric right ventricular outflow tract (RVOT) cardiac anomalies. Adult syngenic Lewis rat SMCs (2 x 10(6)) were seeded onto a new biodegradable copolymer sponge made of epsilon-caprolactone-co-L-lactide reinforced with poly-L-lactide fabric (PCLA). Two weeks after seeding, the patch was used to repair a surgically created RVOT defect in an adult rat. At 8 weeks after implantation the spongy copolymer component was biodegraded, and SM tissue and extracellular matrices containing elastin fibers were present in the scaffolds. By 22 weeks more fibroblasts and collagen were present (p < 0.05). The number of capillaries in the grafts also increased (p < 0.001) between 8 and 22 weeks. The fibrous poly-L-lactide component of the PCLA scaffold remained. The 22-week grafts maintained their thickness and surface area in the RVOT. The SMCs prior to implantation were in a synthetic phenotype and developed in vivo into a more contractile phenotype. By 8 weeks the patches were endothelialized on their endocardial surfaces. Future work to increase the SM tissue and elastin content in the patch will be necessary before implantation into a pediatric large-animal model is tested.


Subject(s)
Absorbable Implants , Biocompatible Materials , Bioprosthesis , Heart Defects, Congenital/surgery , Heart Valve Prosthesis , Myocytes, Smooth Muscle/transplantation , Ventricular Outflow Obstruction/surgery , Animals , Biodegradation, Environmental , Caproates , Culture Techniques , Lactones , Polyesters , Polyglycolic Acid , Rats , Rats, Inbred Lew , Transplants
8.
Circulation ; 109(17): 2058-67, 2004 May 04.
Article in English | MEDLINE | ID: mdl-15078802

ABSTRACT

BACKGROUND: Myocardial ischemia provides a potent stimulus to angiogenesis, and the mobilization and differentiation of endothelial progenitor cells (EPCs) has been shown to be important in this process. An elevated level of C-reactive protein (CRP) has emerged as one of the most powerful predictors of cardiovascular disease. However, the impact of CRP on EPC biology is unknown. METHODS AND RESULTS: EPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in endothelial cell basal medium-2 in the absence and presence of CRP (5 to 20 microg/mL), rosiglitazone (1 micromol/L), and/or vascular endothelial growth factor. EPC differentiation, survival, and function were assayed. CRP at concentrations > or =15 microg/mL significantly reduced EPC cell number, inhibited the expression of the endothelial cell-specific markers Tie-2, EC-lectin, and VE-cadherin, significantly increased EPC apoptosis, and impaired EPC-induced angiogenesis. EPC-induced angiogenesis was dependent on the presence of nitric oxide, and CRP treatment caused a decrease in endothelial nitric oxide synthase mRNA expression by EPCs. However, all of these detrimental CRP-mediated effects on EPCs were attenuated by pretreatment with rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. CONCLUSIONS: Human recombinant CRP, at concentrations known to predict adverse vascular outcomes, directly inhibits EPC differentiation, survival, and function, key components of angiogenesis and the response to chronic ischemia. This occurs in part via an effect of CRP to reduce EPC eNOS expression. The PPARgamma agonist rosiglitazone inhibits the negative effects of CRP on EPC biology. The ability of CRP to inhibit EPC differentiation and survival may represent an important mechanism that further links inflammation to cardiovascular disease.


Subject(s)
C-Reactive Protein/pharmacology , Endothelial Cells/drug effects , Apoptosis/drug effects , C-Reactive Protein/antagonists & inhibitors , Cardiovascular Diseases/etiology , Cell Differentiation/drug effects , Cells, Cultured/drug effects , Endothelial Cells/cytology , Endothelial Cells/physiology , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Humans , Inflammation , Male , Neovascularization, Physiologic/drug effects , Nitric Oxide/biosynthesis , Nitric Oxide/physiology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type III , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/pharmacology , Rosiglitazone , Thiazolidinediones/pharmacology , Transcription Factors/agonists , Transcription Factors/biosynthesis , Transcription Factors/genetics , Vascular Endothelial Growth Factor A/pharmacology
9.
J Thorac Cardiovasc Surg ; 127(4): 1180-7, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15052220

ABSTRACT

OBJECTIVE: We evaluated the effect of transplanted cell type, time, and region of the heart on transgene expression to determine the potential of combined gene and cell delivery for myocardial repair. METHODS: Lewis rats underwent myocardial cryoinjury 3 weeks before transplantation with heart cells (a mixed culture of cardiomyocytes, smooth muscle cells, endothelial cells and fibroblasts, n = 13), vascular endothelial growth factor-transfected heart cells (n = 13), skeletal myoblasts (n = 13), vascular endothelial growth factor-transfected skeletal myoblasts (n = 13), or medium (control, n = 12). Vascular endothelial growth factor expression in the scar, border zone, and normal myocardium was evaluated at 3 days and at 1, 2, and 4 weeks by means of quantitative polymerase chain reaction. Transplanted cells and vascular endothelial growth factor protein were identified immunohistologically on myocardial sections. RESULTS: Vascular endothelial growth factor levels were very low in control scars but increased transiently after medium injection. Transplantation with heart cells and skeletal myoblasts significantly increased vascular endothelial growth factor expression in the scar and border zone. Transplantation of vascular endothelial growth factor-transfected heart cells and vascular endothelial growth factor-transfected skeletal myoblasts further augmented vascular endothelial growth factor expression, resulting in 4- to 5-fold greater expression of vascular endothelial growth factor in the scar at 1 week. Peak vascular endothelial growth factor expression was greater and earlier in vascular endothelial growth factor-transfected heart cells than in vascular endothelial growth factor-transfected skeletal myoblasts. Vascular endothelial growth factor was primarily expressed by the transplanted cells. Some of the transplanted heart cells and vascular endothelial growth factor-transfected heart cells were identified in the endothelial layer of blood vessels in the scar. CONCLUSIONS: Transplantation of heart cells and skeletal myoblasts induces vascular endothelial growth factor expression in myocardial scars and is greatly augmented by prior transfection with a vascular endothelial growth factor transgene. Vascular endothelial growth factor expression is limited to the scar and border zone for 4 weeks. Both heart cells and skeletal myoblasts may be excellent delivery vehicles for cell-based myocardial gene therapy.


Subject(s)
Cell Transplantation , Gene Expression Regulation/genetics , Myocytes, Cardiac/transplantation , Transgenes/genetics , Vascular Endothelial Growth Factor A/genetics , Animals , Disease Models, Animal , Female , Immunohistochemistry , Male , Models, Cardiovascular , Myoblasts, Skeletal/metabolism , Myoblasts, Skeletal/transplantation , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/therapy , Myocytes, Cardiac/metabolism , Neovascularization, Physiologic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred Lew , Transfection , Vascular Endothelial Growth Factor A/biosynthesis
10.
Circulation ; 109(7): 833-6, 2004 Feb 24.
Article in English | MEDLINE | ID: mdl-14967730

ABSTRACT

BACKGROUND: Because complement-mediated vascular injury participates in atherosclerosis and C-reactive protein (CRP) can activate the complement cascade, we sought to determine whether CRP affects the expression of the protective complement-inhibitory factors on the cell surface of endothelial cells (ECs). METHODS AND RESULTS: Human coronary artery or human saphenous vein ECs were incubated with CRP (0 to 100 microg/mL, 0 to 72 hours), and the expression of the complement-inhibitory proteins decay-accelerating factor (DAF), membrane cofactor protein (CD46), and CD59 were measured by flow cytometry. Incubation with CRP resulted in a significant increase in the expression of all 3 proteins. CRP-induced upregulation of DAF required increased steady-state mRNA and de novo protein synthesis. The increased expression of complement-inhibitory proteins was functionally effective, resulting in significant reduction of complement-mediated lysis of antibody-coated human saphenous vein ECs. CONCLUSIONS: These observations provide evidence for a possible protective role for CRP in atherogenesis.


Subject(s)
Antigens, CD/biosynthesis , Arteriosclerosis/prevention & control , C-Reactive Protein/physiology , CD55 Antigens/biosynthesis , CD59 Antigens/biosynthesis , Endothelium, Vascular/drug effects , Membrane Glycoproteins/biosynthesis , Antibody-Dependent Cell Cytotoxicity/drug effects , Antigens, CD/genetics , Arteriosclerosis/metabolism , C-Reactive Protein/pharmacology , CD55 Antigens/genetics , CD59 Antigens/genetics , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Coronary Vessels/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Gene Expression Regulation/drug effects , Humans , Membrane Cofactor Protein , Membrane Glycoproteins/genetics , RNA, Messenger/biosynthesis , Saphenous Vein/cytology
11.
Cell Transplant ; 13(2): 169-177, 2004 Mar.
Article in English | MEDLINE | ID: mdl-28853932

ABSTRACT

Autologous smooth muscle cell (SMC)-seeded biodegradable scaffolds could be a suitable material to repair some pediatric right ventricular outflow tract (RVOT) cardiac anomalies. Adult syngenic Lewis rat SMCs (2 × 106) were seeded onto a new biodegradable copolymer sponge made of ∊-caprolactone-co-L-lactide reinforced with poly-L-lactide fabric (PCLA). Two weeks after seeding, the patch was used to repair a surgically created RVOT defect in an adult rat. At 8 weeks after implantation the spongy copolymer component was biodegraded, and SM tissue and extracellular matrices containing elastin fibers were present in the scaffolds. By 22 weeks more fibroblasts and collagen were present (p < 0.05). The number of capillaries in the grafts also increased (p < 0.001) between 8 and 22 weeks. The fibrous poly-L-lactide component of the PCLA scaffold remained. The 22-week grafts maintained their thickness and surface area in the RVOT. The SMCs prior to implantation were in a synthetic phenotype and developed in vivo into a more contractile phenotype. By 8 weeks the patches were endothelialized on their endocardial surfaces. Future work to increase the SM tissue and elastin content in the patch will be necessary before implantation into a pediatric large-animal model is tested.

12.
J Thorac Cardiovasc Surg ; 126(6): 1886-91, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14688701

ABSTRACT

OBJECTIVES: Elevated levels of C-reactive protein are one of the strongest prognostic factors in atherosclerosis. In addition to predicting vascular disease, C-reactive protein may directly facilitate the development of a proinflammatory and proatherosclerotic phenotype. Recent studies have demonstrated marked up-regulation of various adhesion molecules and inflammatory responses in endothelial cells subjected to C-reactive protein. The nuclear factor-kappaB signal transduction is known to play a key role in the expression of these proatherogenic entities. This study examines the direct effects of C-reactive protein on nuclear factor-kappaB activation and related mechanisms in saphenous vein endothelial cells. METHODS: The activation of nuclear factor-kappaB was determined by confocal microscopy assessing the nuclear localization of nuclear factor-kappaB in endothelial cells incubated with C-reactive protein (50 microg/mL) for 30 minutes and 3 hours. Cells not incubated with C-reactive protein were used as negative controls, and cells incubated with tumor necrosis factor-alpha (10 ng/mL) for 15 minutes were used as positive controls in all studies. The degradation of IkappaB-alpha and IkappaB-beta was assessed by Western blotting of the cell lysates obtained from cells incubated with human recombinant C-reactive protein (50 microg/mL) for 15 minutes, 30 minutes, and 1 hour. RESULTS: Nuclear factor-kappaB nuclear translocation in endothelial cells increased significantly after 30 minutes of incubation with C-reactive protein (P <.01). Nuclear localization of nuclear factor-kappaB returned to baseline levels after 3 hours of incubation with C-reactive protein. Incubation with C-reactive protein resulted in degradation of IkappaB-alpha that was maximal at 30 minutes (P <.05). C-reactive protein showed no significant effect on IkappaB-beta degradation. CONCLUSIONS: These data demonstrate, for the first time, that C-reactive protein activates the nuclear factor-kappaB signal transduction pathway in endothelial cells. Degradation of IkappaB-alpha, but not IkappaB-beta, seems to be the major pathway leading to nuclear factor-kappaB nuclear translocation and activation induced by C-reactive protein. These data support the concept that C-reactive protein, at concentrations known to predict diverse vascular insults, directly facilitates a proinflammatory and proatherosclerotic phenotype through activation of nuclear factor-kappaB. These data have important implications for saphenous vein atherosclerosis in patients with elevated C-reactive protein levels.


Subject(s)
Arteriosclerosis/metabolism , C-Reactive Protein/pharmacology , Endothelium, Vascular/metabolism , NF-kappa B/metabolism , Saphenous Vein/metabolism , Signal Transduction , Arteriosclerosis/physiopathology , Blotting, Western , C-Reactive Protein/physiology , Cells, Cultured , Histocytochemistry , Humans , I-kappa B Proteins/metabolism , Microscopy, Confocal , Recombinant Proteins/pharmacology , Signal Transduction/drug effects
13.
J Thorac Cardiovasc Surg ; 126(5): 1537-48, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14666030

ABSTRACT

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.


Subject(s)
Cardiomyopathy, Dilated/surgery , Cryopreservation/methods , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/transplantation , Ventricular Function, Left/physiology , Ventricular Remodeling/physiology , Analysis of Variance , Animals , Cardiac Volume , Cell Division , Cell Transplantation/methods , Cricetinae , Disease Models, Animal , Heart Function Tests , Immunohistochemistry , Male , Muscle, Skeletal/cytology , Muscle, Skeletal/pathology , Muscle, Skeletal/transplantation , Muscle, Smooth, Vascular/cytology , Photomicrography , Probability , Reference Values , Sensitivity and Specificity , Transplantation, Autologous
14.
Ann Thorac Surg ; 76(6): 2062-70; discussion 2070, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14667643

ABSTRACT

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.


Subject(s)
Cell Transplantation , Myocardial Infarction/therapy , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Aorta/cytology , Cardiac Output, Low/etiology , Cardiac Output, Low/prevention & control , Cells, Cultured , Cicatrix/metabolism , Cryopreservation , Elastin/analysis , Enalapril/pharmacology , Heart Injuries/complications , Heart Injuries/therapy , Heart Ventricles/cytology , Heart Ventricles/metabolism , Immunohistochemistry , Muscle, Skeletal/cytology , Muscle, Smooth, Vascular/cytology , Myocardial Infarction/complications , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Myocardium/chemistry , Rats , Rats, Inbred Lew , Ventricular Function, Left
15.
Circulation ; 108 Suppl 1: II219-25, 2003 Sep 09.
Article in English | MEDLINE | ID: mdl-12970236

ABSTRACT

BACKGROUND: Recurrent ventricular dilatation can occur after surgical repair of a left ventricular (LV) aneurysm. Use of an autologous bioengineered muscle graft to replace resected scar tissue may prevent recurrent dilatation and improve cardiac function. METHODS: Vascular smooth muscle cells (SMCs, 5 x 10(6) cells) from rat aortas were seeded onto synthetic PCLA (sponge polymer of epsilon-caprolactone-co-L-lactide reinforced with knitted poly-L-lactide fabric) patches and cultured for 2 weeks to allow tissue formation. Syngenic rats underwent proximal left coronary artery ligation to create a transmural myocardial scar. Four weeks after coronary ligation, cell-seeded patches (n=15) or unseeded patches (n=12) were used for a modified endoventricular circular patch plasty (EVCPP) repair of the infarct area. Ligated controls (n=14) and nonligated normal rats (n=10) had sham surgeries without EVCPP. Cardiac function was assessed by echocardiography and isolated Langendorff heart perfusion. Graft histology and morphology was also assessed. RESULTS: After 8 weeks in vivo, seeded patches were thicker (P<0.05) and smaller in area (P<0.003) than unseeded patches. Only seeded patches had prominent elastic tissue formation (P<0.001) in association with SMCs. LV systolic function by echocardiography was improved in the seeded group compared with both unseeded (P<0.002) and control groups (P<0.0001). LV volumes in both patch repair groups were comparable but were significantly smaller (P<0.05) than controls. LV distensibility tended toward improvement in the seeded group as compared with unseeded hearts, but the difference did not achieve statistical significance (P=0.06). CONCLUSIONS: Surgical repair with muscle-cell seeded grafts reduced abnormal chamber distensibility and improved LV function after myocardial infarction as compared with unseeded grafts. Bioengineered muscle grafts may be superior to synthetic materials for the surgical repair of LV scar.


Subject(s)
Heart Aneurysm/surgery , Muscle, Smooth, Vascular/transplantation , Animals , Cells, Cultured , Heart Aneurysm/pathology , Heart Aneurysm/physiopathology , Male , Myocardial Infarction/mortality , Myocardial Infarction/pathology , Rats , Rats, Inbred Lew , Tissue Engineering , Ventricular Function, Left , Ventricular Remodeling
16.
Semin Thorac Cardiovasc Surg ; 15(3): 277-86, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12973705

ABSTRACT

Cell transplantation and the creation of bioengineered cardiovascular tissues are novel biologic approaches to restore and regenerate failing myocardium. These rapidly evolving therapies may complement and enhance other mechanical and surgical interventions for patients with congestive heart failure, providing cardiac surgeons with a wider range of treatments for patients at risk of congestive heart failure. Proof-of-concept studies have been performed in several experimental animal models of human cardiovascular disease, such as myocardial infarction and dilated cardiomyopathy. Although the exact mechanisms are unclear, cell transplantation restores cardiac function and limits ventricular dilatation. Clinical cell transplantation has been performed in a limited number of patients with encouraging preliminary results. In contrast, bioengineered muscle grafting is largely experimental but offers the promise of myocardial regeneration by replacing irreversibly damaged myocardium with healthy autologous tissue to facilitate more extensive ventricular remodeling surgery.


Subject(s)
Cell Transplantation/methods , Heart Failure/pathology , Heart Failure/therapy , Myocardium/pathology , Tissue Engineering/methods , Animals , Humans , Myoblasts/pathology , Myoblasts/transplantation , Myocardial Infarction/therapy , Myocardial Ischemia/therapy
17.
Circulation ; 108(6): 736-40, 2003 Aug 12.
Article in English | MEDLINE | ID: mdl-12874180

ABSTRACT

BACKGROUND: Adipocyte-derived hormones may represent a mechanism linking insulin resistance to cardiovascular disease. In the present study, we evaluated the direct effects of resistin, a novel adipocyte-derived hormone, on endothelial activation. METHODS AND RESULTS: Endothelial cells (ECs) were incubated with human recombinant resistin (10 to 100 ng/ML, 24 hours), and endothelin-1 (ET-1) release, ET-1 mRNA expression, and nitric oxide (NO) production were assessed. Transient transfection assays were used to evaluate the effects of resistin on transcription of human ET-1 gene promoter. Furthermore, the effects of resistin on AP-1-mutated ET-1 promoter were evaluated. The effects of resistin on expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemoattractant chemokine (MCP-1) were studied in addition to CD40 receptor, CD40 ligand-induced MCP-1 expression, and tumor necrosis factor receptor-associated factor-3 (TRAF3), an inhibitor of CD40 signaling. Incubation of ECs with resistin resulted in an increase in ET-1 release and ET-1 mRNA expression, with no change in NO production. Whereas treatment with resistin resulted in an increase in ET-1 promoter activity, the AP-1-mutated promoter was inactive after resistin stimulation. Additionally, resistin-treated cells showed increased expression of VCAM-1 and MCP-1, with concomitant reductions in TRAF-3 expression. Resistin did not alter CD40 receptor expression; however, increased CD40 ligand induced MCP-1 production. CONCLUSIONS: The novel adipokine resistin exerts direct effects to promote EC activation by promoting ET-1 release, in part by inducing ET-1 promoter activity via the AP-1 site. Furthermore, resistin upregulates adhesion molecules and chemokines and downregulates TRAF-3, an inhibitor of CD40 ligand signaling. In this fashion, resistin may be mechanistically linked to cardiovascular disease in the metabolic syndrome.


Subject(s)
Adipocytes/metabolism , Endothelin-1/genetics , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Hormones, Ectopic/pharmacology , Intercellular Signaling Peptides and Proteins , CD40 Ligand/pharmacology , Cell Communication/physiology , Cells, Cultured , Chemokine CCL2/metabolism , Endothelin-1/metabolism , Endothelium, Vascular/cytology , Hormones, Ectopic/genetics , Humans , Nitric Oxide/metabolism , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/physiology , Proteins/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/pharmacology , Resistin , TNF Receptor-Associated Factor 3 , Transfection , Vascular Cell Adhesion Molecule-1/metabolism
18.
Mol Cell Biochem ; 247(1-2): 101-9, 2003 May.
Article in English | MEDLINE | ID: mdl-12841637

ABSTRACT

The endothelial layer is a key component of the cardiovascular system. Recent evidence indicates that strategies aimed at preserving the endothelium may have important implications in the battle against cardiovascular disease. Nitric oxide remains the critical factor determinant of endothelial function. Understanding the regulatory components involved in nitric oxide production may elucidate novel targets for improving compromised vascular function. The caveolae/caveolin system has recently become of interest due to its ability to regulate endothelial nitric oxide synthase activity. The caveolae/caveolin system is a multifaceted structure in the plasma membrane, which plays an integral role in cellular signaling. Recognizing the potential of this specialized domain may provide the fundamental knowledge to target the endothelium in disease.


Subject(s)
Caveolins/physiology , Nitric Oxide/metabolism , Nitric Oxide/pharmacokinetics , Animals , Biological Availability , Calcium/metabolism , Caveolae/pathology , Caveolae/physiology , Caveolin 1 , Health , Humans , Molecular Chaperones/metabolism , Phosphorylation , Signal Transduction
19.
Eur J Cardiothorac Surg ; 23(6): 907-16, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12829066

ABSTRACT

Current therapies for congestive heart failure are limited in efficacy or in applicability. Cardiac cell transplantation offers a novel therapeutic approach to improve heart function. Although significant progress has been made over the past decade in the development of cell transplantation, only recently have investigators studied the changes in ventricular function following cell transplantation. This review article describes the latest research developments, evaluates recent studies of ventricular function after cell transplantation, and discusses the future directions of cell transplantation as a new therapy to 'repair broken hearts'.


Subject(s)
Heart Failure/surgery , Myocytes, Cardiac/transplantation , Ventricular Dysfunction/surgery , Animals , Bone Marrow Transplantation , Echocardiography , Electric Conductivity , Heart Failure/diagnosis , Heart Failure/etiology , Models, Animal , Myocardial Ischemia/complications , Myocardial Ischemia/diagnosis , Perfusion , Tomography, Emission-Computed, Single-Photon , Ventricular Dysfunction/diagnosis , Ventricular Dysfunction/etiology
20.
Circulation ; 107(13): 1783-90, 2003 Apr 08.
Article in English | MEDLINE | ID: mdl-12665485

ABSTRACT

BACKGROUND: Accumulating evidence suggests that C-reactive protein (CRP), in addition to predicting vascular disease, may actively facilitate lesion formation by inciting endothelial cell activation. Given the central importance of angiotensin type 1 receptor (AT1-R) in the pathogenesis of atherosclerosis, we examined the effects of CRP on AT1-R expression and kinetics in vascular smooth muscle (VSM) cells. In addition, the effects of CRP on VSM migration, proliferation, and reactive oxygen species (ROS) production were evaluated in the presence and absence of the angiotensin receptor blocker, losartan. Lastly, the effects of CRP (and losartan) on neointimal formation were examined in vivo in a rat carotid angioplasty model. METHODS AND RESULTS: The effects of human recombinant CRP (0 to 100 microg/mL) on AT1-R transcript, mRNA stability, and protein expression were studied in cultured human VSM cells. AT1-R binding was assessed with 125I-labeled angiotensin II (Ang II). VSM migration was assessed with wound cell migration assays, whereas VSM proliferation was determined with [3H]-incorporation and cell number. The effects of CRP (and losartan) on Ang II-induced ROS production were evaluated by 2',7'-dichlorofluorescein fluorescence. Lastly, the effects of CRP (and losartan) on neointimal formation, VSM cell migration, proliferation, and matrix formation were studied in vivo in a rat carotid artery balloon injury model. CRP markedly upregulated AT1-R mRNA and protein expression and increased AT1-R number on VSM cells. CRP promoted VSM migration and proliferation in vitro and increased ROS production. Furthermore, CRP potentiated the effects of Ang II on these processes. In the rat carotid artery angioplasty model, exposure to CRP resulted in an increase in cell migration and proliferation, collagen and elastin content, and AT1-R expression, as well as an increase in neointimal formation; these effects were attenuated by losartan. CONCLUSIONS: CRP, at concentrations known to predict cardiovascular events, upregulates AT1-R-mediated atherosclerotic events in vascular smooth muscle in vitro and in vivo. These data lend credence to the notion that CRP functions as a proatherosclerotic factor as well as a powerful risk marker.


Subject(s)
C-Reactive Protein/pharmacology , Muscle, Smooth, Vascular/metabolism , Receptors, Angiotensin/biosynthesis , Up-Regulation , Angioplasty, Balloon/adverse effects , Animals , Carotid Stenosis/etiology , Carotid Stenosis/pathology , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Gene Expression Regulation , Humans , Kinetics , Male , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/analysis , Receptor, Angiotensin, Type 1 , Receptors, Angiotensin/genetics , Receptors, Angiotensin/metabolism
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