RESUMEN

BACKGROUND: Stem cell therapy has been shown to attenuate the reduction of left ventricular function following myocardial infarction. Most studies have utilized either a direct injection or intra-coronary infusion of cells, but cytokine mobilization of stem cells in the murine model of acute myocardial infarction has been reported to induce similar improvement in cardiac function. METHODS: An antero-apical infarction was induced in swine by balloon occlusion, followed by the daily administration of granulocyte colony stimulating factor (G-CSF) or placebo for 5 days. We used left ventricular angiograms and 2D echocardiograms to assess global function, and 3D echocardiograms to assess regional function prior to infarction, immediately following infarction, and at 8 weeks. Histologic evaluation was performed after sacrifice at 8 weeks. RESULTS: There was no significant difference in early or late post-infarction left ventricular ejection fraction or in myocardial histology between the two groups. Following G-CSF therapy, however, 3D echocardiography demonstrated that the regional ejection fractions of the infarcted segments showed a 50.3% improvement in the G-CSF pigs compared to a 7.4% deterioration in the untreated pigs (p=0.005). CONCLUSIONS: Global left ventricular ejection fraction remained unchanged, and there is no histologic evidence for infarct attenuation following G-CSF infusion in the porcine infarct-reperfusion model. There was recovery of regional function in the infarcted segment in the G-CSF pigs. These data suggest that bone marrow mobilization in larger species has limited potential as a therapy designed to replace infarcted myocardium or to improve overall cardiac function, although further studies are needed to examine regional effect in the infarct area.

Asunto(s)

Factor Estimulante de Colonias de Granulocitos/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Infarto del Miocardio/fisiopatología , Animales , Colágeno/metabolismo , Angiografía Coronaria , Ecocardiografía Tridimensional , Ventrículos Cardíacos , Recuento de Leucocitos , Infarto del Miocardio/diagnóstico , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Volumen Sistólico/efectos de los fármacos , Porcinos

RESUMEN

UNLABELLED: Direct intramyocardial injection of mesenchymal stem cells (MSCs) improves left ventricular ejection fraction (LVEF) and may increase ventricular arrhythmia in hearts with myocardial infarction (MI). We hypothesized that intravenous MSCs given early after acute MI would engraft in injured myocardium, improve LV function, and result in pro-arrhythmic electrical remodeling. We created an apical infarction in swine by balloon occlusion/reperfusion, administered diI-labeled allogeneic bone marrow derived MSCs intravenously 30 min post-reperfusion and measured LVEF and wall thickness at baseline, 1 month, and 3 months. Epicardial effective refractory periods (ERPs) were determined before sacrifice. At 3 months, treated pigs [n=7] had significantly higher LVEF than controls [n=8] (49+/-2% vs. 44+/-3%, P=0.015) and significantly less wall thickening of non-infarcted myocardium. ERPs were significantly shorter than controls at all pacing cycle lengths (P

Asunto(s)

Trasplante de Células Madre Mesenquimatosas/métodos , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Función Ventricular Izquierda/fisiología , Animales , Técnicas de Cultivo de Célula , Angiografía Coronaria , Modelos Animales de Enfermedad , Ecocardiografía , Electrofisiología/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/patología , Infarto del Miocardio/diagnóstico por imagen , Reperfusión , Porcinos

RESUMEN

BACKGROUND: We investigated the efficacy of directly injected allogenic bone marrow-derived mesenchymal stem cells in improving left ventricular function in a porcine model of myocardial infarction. METHODS: Left ventricular infarction was created in 16 adult Yorkshire pigs by coil embolization and thrombotic occlusion distal to the second diagonal artery. One month after myocardial infarction was induced, the animals were randomized to either direct injection of allogenic mesenchymal stem cells or sham treatment (culture medium). Allogenic bromodeoxyuridine-labeled mesenchymal stem cells (2 +/- 0.1 x 10(8)) were directly injected into the infarct and peri-infarct areas during an open chest procedure. No immunosuppressive therapy was used. The left ventricular function was measured using serial biplane left ventricular angiography at baseline, 30, 60, and 90 days before sacrifice. Mesenchymal stem cells were localized using bromodeoxyuridine, and differentiation of mesenchymal stem cells was assessed by confocal microscopic colocalization of bromodeoxyuridine with immunofluorescent antibodies specific for cardiomyocytes (troponin I and MF-20) and endothelial cells (von Willebrand factor). RESULTS: Mesenchymal stem cells labeled with bromodeoxyuridine engrafted the peri-infarct zone and colocalized with both cardiomyocyte-specific and endothelial cell-specific immunofluorescence. No intramyocardial bromodeoxyuridine was observed in sham-treated animals. At the time of the mesenchymal stem cell injection 30 days after myocardial infarction, the left ventricular ejection fraction (LVEF) was 58% +/- 3% in mesenchymal stem cell-treated pigs and 56% +/- 2% in sham-treated pigs (P = NS). LVEF deteriorated progressively thereafter in untreated pigs (8.5% and 10.5% decline at 60 days and 90 days after myocardial infarction, respectively), but was preserved in mesenchymal stem cell-treated pigs (2.1% increase and -2.0% decline at 60 and 90 days post-MI respectively) (P < .05). CONCLUSIONS: Direct intramyocardial injection of mesenchymal stem cells results in successful intramyocardial engraftment and differentiation into cardiomyocytes and endothelial cells and preserves left ventricular function after myocardial infarction in pigs.

Asunto(s)

Trasplante de Células Madre Mesenquimatosas , Infarto del Miocardio/terapia , Disfunción Ventricular Izquierda/terapia , Animales , Antígenos de Diferenciación , Diferenciación Celular , Modelos Animales de Enfermedad , Células Endoteliales , Inmunohistoquímica , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos , Volumen Sistólico , Porcinos , Factores de Tiempo , Trasplante Homólogo , Disfunción Ventricular Izquierda/fisiopatología

RESUMEN

PURPOSE: Tissue engineering has been used for bladder augmentations with small intestinal submucosa (SIS). Although favorable short-term outcomes have been reported, long-term followup has been poor. We investigate whether tissue engineering with stem cells improves the morphological and genetic composition. MATERIALS AND METHODS: A total of 33 Lewis rats (Harlan Laboratories, Indianapolis, Indiana) were used to investigate bladder augmentations with 4-layer SIS in certain groups, including the control group (sham operation), partial cystectomy with oversewn defect group (OG), augmentation with unseeded SIS group (USG) and augmentation with stem cell seeded SIS group (SSG). Bladders from 4 rats per group were harvested 1 and 3 months after surgery. Morphological analyses were performed using Masson's trichrome and immunohistochemical staining with cytokeratin AE1/AE3, smooth muscle alpha-actin and S100. Gene expression was evaluated using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for collagen I (CI), collagen III (CIII), cytokeratins 8 and 19, and smooth muscle myosin heavy chain (MHC). RESULTS: At 1 month trichrome staining revealed collagen admixed with indiscrete cells and morphology similar to that in controls in USG and SSG, respectively. Discrete smooth muscles fascicles and S100 staining were found in all groups except USG. Organized urothelium with increased basal cell layer staining was present in controls and SSG only. At 3 months increased collagen formation was present in OG and USG. Immunostaining showed hyperplasia of the urothelium with increased staining of the basal cell layer, discrete muscle fascicles and positive nerve staining in all groups. Using quantitative RT-PCR expression levels in SSG were more improved than in USG, especially for CI, CIII and MHC. This was further evident at 3 months when CI and CIII were over expressed in OG and USG but not in the control group or SSG. Furthermore, RT-PCR showed that cytokeratins 8 and 19, and MHC had greater expression levels in SSG than in USG. CONCLUSIONS: Bladder reconstitution occurs more rapidly using stem cell seeded SIS. Although in USG and SSG all 3 cellular constituents appear to develop by 3 months, only SSG had gene expression levels similar to those in controls. The results suggest an explanation for the fibrosis noted in unseeded SIS bladder augmentations and the possible solution using stem cells.

Asunto(s)

Trasplante de Médula Ósea , Mucosa Intestinal , Ingeniería de Tejidos/métodos , Vejiga Urinaria/cirugía , Animales , Intestino Delgado , Ratas , Ratas Endogámicas Lew , Vejiga Urinaria/anatomía & histología

RESUMEN

Increased nuclear factor kappaB (NF-kappaB) activity is associated with increased tumor cell survival in multiple myeloma. The function of NF-kappaB is inhibited through binding to its inhibitor, IkappaB. Release of activated NF-kappaB follows proteasome-mediated degradation of IkappaB resulting from phosphorylation of the inhibitor and, finally, conjugation with ubiquitin. We report that myeloma cells have enhanced IkappaBalpha phosphorylation and increased NF-kappaB activity compared with normal hematopoietic cells. The proteasome inhibitor PS-341 blocked nuclear translocation of NF-kappaB, blocked NF-kappaB DNA binding, and demonstrated consistent antitumor activity against chemoresistant and chemosensitive myeloma cells. The sensitivity of chemoresistant myeloma cells to chemotherapeutic agents was markedly increased (100,000-1,000,000-fold) when combined with a noncytotoxic dose of PS-341 without affecting normal hematopoietic cells. Similar effects were observed using a dominant negative super-repressor for IkappaBalpha. Thus, these results suggest that inhibition of NF-kappaB with PS-341 may overcome chemoresistance and allow doses of chemotherapeutic agents to be markedly reduced with antitumor effects without significant toxicity.

Asunto(s)

Ácidos Borónicos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Mieloma Múltiple/metabolismo , Pirazinas/farmacología , Transporte Activo de Núcleo Celular , Adenoviridae/genética , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Bortezomib , División Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Cisteína Endopeptidasas , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Genes Dominantes , Humanos , Proteínas I-kappa B/metabolismo , Melfalán/farmacología , Microscopía Fluorescente , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Ubiquitina/metabolismo

RESUMEN

When NF-kappaB proteins are bound to IkappaBalpha, they remain in the cytosol, and are unable to act as transcription factors. Phosphorylation of IkappaBalpha at Serine32 and Serine36 has been shown to stimulate ubiquitination followed by proteasome-mediated degradation of IkappaBalpha, resulting in the release of active NF-kappaB. NF-kappaB activity is associated with bone loss and B cell growth as well as chemotherapy resistance. Because previous studies have shown abnormalities of the IkappaBalpha gene in patients with lymphoma, we determined whether alterations of this gene also occur in multiple myeloma (MM). We determined the DNA sequence of the IkappaBalpha gene from bone marrow mononuclear cells from 18 MM patients and 24 healthy subjects as well as two MM cell-lines. We identified eight polymorphisms. Statistically, the prevalence of three polymorphisms, one in exon 1 and two in exon 6, were significantly higher in MM patients (alpha>1) compared with samples from control subjects. Six of eight polymorphisms in myeloma samples have also been identified in previous studies of IkappaBalpha sequences derived from lymphoma samples. In addition, we detected two polymorphisms in the IkappaBalpha gene that have not been previously reported. Together, these results provide the basis for future evaluation the IkappaBalpha/NF-kappaB pathway in MM patients.

Asunto(s)

Cromosomas Humanos Par 14 , Proteínas de Unión al ADN/genética , Proteínas I-kappa B , Mieloma Múltiple/genética , FN-kappa B/antagonistas & inhibidores , Polimorfismo Genético , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN , Exones , Humanos , Mieloma Múltiple/epidemiología , Inhibidor NF-kappaB alfa , Reacción en Cadena de la Polimerasa , Factores de Riesgo

RESUMEN

OBJECTIVE: The human insulin-like growth factor 2 (IGF2) gene was thought to be imprinted and expressed only from the paternal allele in normal tissue. MATERIALS AND METHODS: Initially, we analyzed the imprinting status of IGF2 in bone marrow cells from 49 patients with myelodysplastic syndromes (MDS) utilizing the Apa I polymorphism of IGF2. Thirteen bone marrow and 14 peripheral blood samples from normal individuals served as controls. We utilized normal peripheral blood T lymphocytes to examine the relationship between genomic imprinting and cell proliferation. Expression of IGF2 was quantified by real-time PCR and proliferation of T cells was measured by 3H-thymidine incorporation. Furthermore, methylation status of the imprinting controlling region (ICR) was analyzed by subcloning and sequencing of genomic DNA after sodium bisulfite modification. RESULTS: Among 24 patients who were heterozygous for IGF2, loss of imprinting (LOI) occurred in 22 cases (92%). Surprisingly, LOI of IGF2 occurred in the normal bone marrow cells, but the normal peripheral blood cells showed retention of imprinting (ROI). Unstimulated normal T cells showed ROI. After 24 hours of exposure to PHA, these cells changed their IGF2 imprinting status from ROI to LOI. Concomitantly, their IGF2 RNA levels increased up to sixfold and their proliferation increased 10- to 20-fold. In contrast, normal T cells not stimulated with PHA did not develop LOI of IGF2, had negligible levels of IGF2 RNA, and did not increase their proliferation. In unstimulated T cells, the CpG islands of the ICR were completely methylated on one allele and nearly completely unmethylated on the other allele. After PHA stimulation, the CpG islands at the ICR became completely methylated on both alleles. CONCLUSION: LOI of IGF2 is strongly associated with cell proliferation and is not limited to cancer cells.

Asunto(s)

Impresión Genómica , Células Madre Hematopoyéticas/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Estudios de Casos y Controles , División Celular , Islas de CpG/genética , Metilación de ADN , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Síndromes Mielodisplásicos/patología , Polimorfismo Genético , ARN Mensajero/biosíntesis , Linfocitos T/citología , Linfocitos T/metabolismo