RESUMEN
OBJECTIVE: Endothelial progenitor cells (EPCs) participate in vascular homeostasis and angiogenesis. The aim of the present study was to explore EPC number and function in relation to cardiovascular risk, gender, and reproductive state. METHODS AND RESULTS: As measured by flow-cytometry in 210 healthy subjects, CD34(+)KDR(+) EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17beta-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo. CONCLUSIONS: EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.
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Enfermedades Cardiovasculares/etiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Estrógenos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Caracteres Sexuales , Adulto , Anciano , Animales , Ataxina-1 , Ataxinas , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Adhesión Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Estradiol/farmacología , Femenino , Homeostasis/fisiología , Humanos , Recién Nacido , Masculino , Ciclo Menstrual/metabolismo , Persona de Mediana Edad , Neovascularización Fisiológica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Receptores de Estrógenos/metabolismo , Factores de RiesgoRESUMEN
OBJECTIVE: Our purpose was to study in vitro whether phenotypically-distinct interstitial cell clones from bovine aortic valve (BVIC) possess different calcifying potential in response to endotoxin (lipopolysaccharide [LPS]) and phosphate (Pi). METHODS AND RESULTS: Among various clones of BVIC obtained by limited dilution technique we selected 4 clones displaying different growth patterns and immunophenotypes. Uncloned and cloned cells were treated with combinations of LPS (100 ng/mL) and Pi (2.4 mmol/L). Uncloned BVIC showed increased alkaline phosphatase activity (ALP) after treatment with LPS, which resulted in calcification after addition of Pi. Among BVIC clones, only Clone 1 (fibroblast-like phenotype) showed a relevant increase in ALP after LPS treatment in parallel with prevention of smooth muscle (SM) alpha-actin accumulation. No effect was observed in clonal cells harboring a more stable SM cell-like profile (Clone 4). None of the isolated clones calcified but mineralization was induced in the presence of LPS plus Pi when Clone 1 was cocultured with Clone 4 or after seeding on type I collagen sponges. CONCLUSIONS: Endotoxin and phosphate can act as valve calcification promoters by targeting specific fibroblast-like interstitial valve cells that possess a unique procalcific potential.
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Válvula Aórtica/efectos de los fármacos , Válvula Aórtica/patología , Calcinosis/etiología , Enfermedades de las Válvulas Cardíacas/etiología , Lipopolisacáridos/toxicidad , Fosfatos/toxicidad , Fosfatasa Alcalina/metabolismo , Animales , Válvula Aórtica/metabolismo , Calcinosis/metabolismo , Calcinosis/patología , Calcio/metabolismo , Bovinos , Células Clonales , Técnicas de Cocultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , FenotipoRESUMEN
Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 x 10(6)) or AF-MSCs (2 x 10(6)) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 +/- 129,65 cells/mm2 for CMTMR labeling and 1613.18 +/- 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 +/- 156.28 cells/mm2) in comparison with BM-MSCs (1434.50 +/- 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 +/- 74.51/mm2 (AF-MSCs) versus 1420.58 +/- 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 +/- 0.48) than AF-MSCs (10.83 +/- 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 +/- 0.20 for BM-MSCs and 4.16 +/- 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) alpha-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 +/- 18.91 vs. 150.36 +/- 24.08 cells/mm2, p < 0.01) and CMs (417.91 +/- 100.95 vs. 237.43 +/- 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 +/- 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 +/- 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49 +/- 17.37/mm2 for AF-MSCs vs. 148.69 +/- 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole reconstitution.
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Células Madre Adultas/trasplante , Frío/efectos adversos , Células Madre Fetales/trasplante , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Miocardio/patología , Células Madre Adultas/citología , Células Madre Adultas/fisiología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Ecocardiografía , Células Madre Fetales/citología , Células Madre Fetales/fisiología , Corazón/anatomía & histología , Corazón/fisiología , Hemodinámica , Humanos , Inmunofenotipificación , Masculino , Células Madre Mesenquimatosas/citología , Necrosis , Osteogénesis/fisiología , Ratas , Ratas Endogámicas F344 , Ratas DesnudasRESUMEN
BACKGROUND: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs, as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines, were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT). METHODS AND RESULTS: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay, respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT, the levels of both EPC and VEGF/SDF-1 markedly increased (P < .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential, but on DSAT the baseline level was incompletely attained. CONCLUSIONS: In response to SAT, patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential.
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Células Endoteliales/citología , Endotelio/fisiología , Ejercicio Físico/fisiología , Insuficiencia Cardíaca/terapia , Células Madre/citología , Citocinas/biosíntesis , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
The potential of collagen scaffolds for promoting angiogenesis/arteriogenesis was studied in vivo by implantation on healthy or cryoinjured left ventricles of rats up to 60 days post-injury. Blood vessels content and extra-vascular cell infiltration were evaluated within the collagen scaffold, the cryoinjured areas, and the "border zones" of the myocardium facing the cryoinjured zones. The collagen cardiac patches were almost completely absorbed in 60 days and became populated by new arterioles and capillaries in both intact and cryoinjured heart (arterioles in cryoinjured vs. intact zones were about 2,3-fold higher; capillaries in cryoinjured vs. intact zones were 1.7-fold higher). Collagen cardiac patches exerted a "trophic" effect on the organizing granulation tissue that emerged from the wound-healing process, increasing vessel density of 2.7-fold for arterioles and 4-fold for capillaries. Interstitial cells in collagen cardiac patches rarely (<1%) expressed cardiogenic stem cells markers such as Sca-1- or MDR1, whereas markers of neural crest cells GFAP(+)/nestin(+) cells ranged from 3/30% to 30/70% in collagen cardiac patches placed on intact vs. cryoinjured heart, respectively. Myofibroblasts and cardiomyocytes (CM) were absent but macrophages populated the collagen scaffolds even after 60 days from implantation. Western blotting of collagen cardiac patches after implantation on intact/cryoinjured hearts confirmed that markers of endothelial and smooth muscle cells, but not of CM, were expressed. The porous collagen scaffold was able to elicit a powerful angiogenetic and arteriogenetic response in the intact and cryoinjured hearts, representing an ideal tool for therapeutic angio-arteriogenesis and a potentially useful substrate for stem cell seeding.
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Colágeno/farmacología , Lesiones Cardíacas , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/patología , Animales , Colágeno/ultraestructura , Microscopía Electrónica de Rastreo , Fenotipo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: Peripheral arterial disease (PAD) is a threatening complication of diabetes. As endothelial progenitor cells (EPCs) are involved in neovasculogenesis and maintenance of vascular homeostasis, their impairment may have a role in the pathogenesis of diabetic vasculopathy. This study aimed to establish whether number and function of EPCs correlate with PAD severity in type 2 diabetic patients. METHODS AND RESULTS: EPCs were defined by the expression of CD34, CD133 and KDR, and quantified by flow cytometry in 127 diabetic patients with and without PAD. PAD severity has been assessed as carotid atherosclerosis and clinical stage of leg atherosclerosis obliterans. Diabetic patients with PAD displayed a significant 53% reduction in circulating EPCs versus non-PAD patients, and EPC levels were negatively correlated with the degree of carotid stenosis and the stage of leg claudication. Moreover, the clonogenic and adhesion capacity of cultured EPCs were significantly lower in diabetic patients with PAD versus patients without. CONCLUSIONS: This study demonstrates that EPC decrease is related to PAD severity and that EPC function is altered in diabetic subjects with PAD, strengthening the pathogenetic role of EPC dysregulation in diabetic vasculopathy. EPC count may be considered a novel biological marker of peripheral atherosclerosis in diabetes.
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Angiopatías Diabéticas/patología , Angiopatías Diabéticas/fisiopatología , Células Endoteliales/patología , Enfermedades Vasculares Periféricas/patología , Enfermedades Vasculares Periféricas/fisiopatología , Células Madre/patología , Anciano , Arterias , Células Sanguíneas/patología , Estenosis Carotídea/patología , Estenosis Carotídea/fisiopatología , Estudios de Casos y Controles , Adhesión Celular , Recuento de Células , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Diabetes Mellitus Tipo 2 , Femenino , Citometría de Flujo , Humanos , Claudicación Intermitente/patología , Claudicación Intermitente/fisiopatología , Pierna/irrigación sanguínea , Masculino , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: We investigated the potential association between -514 C-->T polymorphism in the promoter of the hepatic lipase gene (LIPC) and the prevalence of inflammatory cells in the plaque of patients with severe carotid artery stenosis. BACKGROUND: This common LIPC polymorphism has been related to the presence of an atherogenic lipoprotein pattern. METHODS: We studied 68 consecutive patients undergoing carotid endarterectomy. The LIPC genotype was determined by polymerase chain reaction. Endarterectomy specimens were examined by immunocytochemistry using monoclonal antibodies for smooth muscle cells, macrophages, or lymphocytes. RESULTS: In 50 of 68 patients who had evidence of previous ipsilateral ischemic events, 36 (72%) were carriers of the CC genotype, whereas only 14 (28%) were carriers of the CT/TT genotype (p = 0.002). Among the 18 patients without evidence of events, the two genotypes were equally distributed (9 vs. 9). The low-density lipoprotein (LDL) particles were denser in CC than in CT/TT genotype carriers (flotation rate: 0.315 +/- 0.025 vs. 0.356 +/- 0.019, p < 0.0005). The CC genotype was associated with an abundance of macrophages (6.7 +/- 3.5 vs. 2.1 +/- 2.1 cells/area unit in the CT/TT group, p < 0.0005) and a reduced number of smooth muscle cells (6.9 +/- 6.2 vs. 14.5 +/- 6.4 in the CT/TT group, p < 0.0005) in the plaque. An inverse relationship was found between LDL buoyancy and the number of macrophages in the plaque (r = -0.639, p < 0.0005). CONCLUSION: We provide evidence, for the first time, that LIPC promoter -514 C-->T polymorphism, by modulating LDL density, significantly affects the number of macrophages in the plaque and possibly affects the occurrence of cerebrovascular events in patients with carotid artery stenosis.
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Estenosis Carotídea/enzimología , Estenosis Carotídea/genética , Citosina/fisiología , Lipasa/genética , Hígado/enzimología , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Tirosina/genética , Anciano , Arterias Carótidas/enzimología , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Estenosis Carotídea/patología , Femenino , Estudios de Seguimiento , Genotipo , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: Transplantation of stem cells in the acute ischemic myocardium (AMI) may play a role in the recovery of cardiac function. Here, we investigated the ability of amniotic fluid-derived mesenchymal cells (AFC) for phenotypic conversion to vascular cells and cardiomyocytes (CM) when autotransplanted in a porcine model of AMI. METHODS: Single AFC preparations were taken from 12 fetuses 3 days before normal delivery. AFC were expanded in vitro and stored separately until animals of the original litter weighed 22-25 kg. A new model of AMI, i.e. 45-min circumflex coronary occlusion followed by wall dissection, was used to assess AFC differentiation potential. CMFDA-labeled AFC were autogenically transplanted in the ischemic area 1 week after AMI induction. Thirty days later, pigs were sacrificed and the phenotypic profile of transplanted AFC was assessed and compared to the corresponding pre-injection pattern. RESULTS: AFC showed in vitro to be of mesenchymal type also expressing markers of 'embryonic stem' cells (SSEA4 and Oct-4), as well as endothelial (von Willebrand factor, VE-cadherin) and smooth muscle (SM alpha-actin, SM22) cells. Thirty days after transplantation, in the survived AFC (5+/-1%) 'embryonic stem' cell markers disappeared and mesenchymal cell markers were down regulated with the exception of smooth muscle and endothelial antigens. No evidence for expression of cardiac troponin I was found. CONCLUSIONS: In the conditions used in this study, AFC were able to transdifferentiate to cells of vascular cell lineages but not to CM. Thus, porcine AFC may require further ex vivo re-programming to be suitable for therapeutic use in AMI.
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Líquido Amniótico/citología , Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas/métodos , Isquemia Miocárdica/terapia , Animales , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocitos Cardíacos/citología , PorcinosRESUMEN
BACKGROUND: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). METHODS: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ET(A)/ET(B) antagonist bosentan (n=6) or the ET(A)-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ET(A) and ET(B) receptor density were also assessed with quantitative autoradiography. RESULTS: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ET(A) receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ET(A) receptors. CONCLUSIONS: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels.
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Angiotensina II , Aorta , Colágeno/metabolismo , Hipertensión/metabolismo , Hipertensión/patología , Músculo Liso Vascular/patología , Análisis de Varianza , Antagonistas de Receptores de Angiotensina , Animales , Animales Modificados Genéticamente , Antihipertensivos/farmacología , Compuestos de Bifenilo/farmacología , Bosentán , Bloqueadores de los Canales de Calcio/farmacología , Compuestos de Dansilo/farmacología , Modelos Animales de Enfermedad , Antagonistas de los Receptores de Endotelina , Endotelina-1 , Inmunohistoquímica , Irbesartán , Masculino , Nifedipino/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/farmacología , Tetrazoles/farmacologíaRESUMEN
BACKGROUND: Fechtner syndrome (FTNS), also known as Alport-like syndrome, is a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Döhle-like leukocyte inclusions, deafness, and cataract. Although it recently was shown that FTNS derives from mutation of MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA), its pathophysiological characteristics remain unknown. METHODS: We studied a large FTNS family in which 10 components carried a missense mutation of MYH9 determining the D1424H substitution. RESULTS: All affected subjects presented with macrothrombocytopenia and leukocyte Döhle-like bodies consisting of macroaggregates of NMMHC-IIA, but only two subjects had major renal problems characterized by proteinuria and renal failure. Electron microscopy showed focal and segmental effacement of podocytes and loss of the interpodocyte slit diaphragm. Immunohistochemistry showed apical localization of NMMHC-IIA in tubular epithelia and less podocyte staining in the two patients, whereas it was diffuse in normal epithelia. Three patients presented with stable microhematuria, and another five patients had no renal lesions, although they carried the same mutation of MYH9. Therefore, MYH9 mutation per se was responsible for platelet and leukocyte abnormalities, whereas additional predisposing conditions and/or environmental factors are necessary for nephropathy, cataract, and deafness. Looking at podocyte components conferring permselectivity properties to the kidney, we characterized the haplotype of podocin and found cosegregation of one specific allele in the two patients with nephrotic syndrome, suggesting a relationship between podocin features and proteinuria. CONCLUSION: Our study indicates a major role for the NMMHC-IIA abnormality in the pathogenesis of leukocyte, platelet, and kidney defects in FTNS. The basic feature in all cases is aggregation and compartmentation of NMMHC-IIA. However, proteinuria and podocyte lesions are the hallmark of nephropathy in patients who develop renal failure, and podocin may have some function in this setting.
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Glomerulonefritis/genética , Glomerulonefritis/patología , Proteínas Motoras Moleculares , Mutación/genética , Cadenas Pesadas de Miosina/genética , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Miosina Tipo IIA no Muscular/genética , Adolescente , Adulto , Plaquetas/química , Niño , Femenino , Haplotipos/genética , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Riñón/química , Riñón/patología , Leucocitos/química , Leucocitos/ultraestructura , Masculino , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Miosina Tipo IIA no Muscular/biosíntesis , SíndromeRESUMEN
BACKGROUND: Heart valve bioprostheses for cardiac valve replacement are fabricated by xeno- or allograft tissues. Decellularization techniques and tissue engineering technologies applied to these tissues might contribute to the reduction in risk of calcification and immune response. Surprisingly, there are few data on the cell phenotypes obtained after cellularizing these naturally-derived biomaterials in comparison to those expressed in the intact valve. METHODS: Aortic valve interstitial cells (VIC) were used to repopulate the corresponding valve leaflets after a novel decellularization procedure based on the use of ionic and nonionic detergents. VIC from leaflet microexplants at the third passage were utilized to repopulate the decellularized leaflets. Intact, decellularized and repopulated valve leaflets and cultured VIC were examined by immunocytochemical procedures with a panel of antibodies to smooth muscle and nonmuscle differentiation antigens. Intact and cellularized leaflets were also investigated with Western blotting and transmission electron microscopy, respectively. RESULTS: Myofibroblasts and smooth muscle cells (SMC) were mostly localized to the ventricularis of the leaflet whereas fibroblasts were dispersed unevenly. Cultured VIC were comprised of myofibroblasts and fibroblasts with no evidence of endothelial cells and SMC. Two weeks after VIC seeding into decellularized leaflets, grafted cells were found penetrating the bioscaffold. The immunophenotypic and ultrastructural properties of the grafted cells indicated that a VIC heterogeneous mesenchymal cell population was present: fibroblasts, myofibroblasts, SMC, and endothelial cells. CONCLUSIONS: VIC seeding on detergent-treated valve bioscaffolds has the cellular potential to reconstruct a viable aortic valve.
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Válvula Aórtica/citología , Bioprótesis , Técnicas Citológicas , Animales , Western Blotting , Fibroblastos/citología , Inmunohistoquímica , Microscopía Electrónica , Músculo Liso/citología , Porcinos , Ingeniería de Tejidos , Trasplante Heterólogo , Trasplante HomólogoAsunto(s)
Diabetes Mellitus Tipo 2/fisiopatología , Retinopatía Diabética/patología , Enfermedades Vasculares Periféricas/patología , Células Madre/fisiología , Adulto , Anciano , Antígenos CD34/análisis , Diferenciación Celular/fisiología , Retinopatía Diabética/inmunología , Retinopatía Diabética/fisiopatología , Células Endoteliales/inmunología , Endotelio Vascular/citología , Endotelio Vascular/fisiopatología , Humanos , Persona de Mediana Edad , Neovascularización Patológica , Enfermedades Vasculares Periféricas/inmunología , Enfermedades Vasculares Periféricas/fisiopatología , Estudios ProspectivosRESUMEN
Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.
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Líquido Amniótico/citología , Diferenciación Celular , Miocitos Cardíacos/citología , Células Madre/citología , Animales , Antígenos de Diferenciación/metabolismo , Separación Celular/métodos , Transdiferenciación Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Rechazo de Injerto , Proteínas Fluorescentes Verdes/metabolismo , Daño por Reperfusión Miocárdica/terapia , Miocardio/patología , Miocitos Cardíacos/fisiología , Cadenas Pesadas de Miosina/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual , Trasplante de Células Madre , Células Madre/fisiología , Trasplante Homólogo , Troponina I/metabolismoRESUMEN
Different vertebrate species have different cardiac regeneration rates: high in teleost fish, moderate in urodele amphibians, and almost negligible in mammals. Regeneration may occur through stem and progenitor cell differentiation or via dedifferentiation with residual cardiomyocytes reentering the cell cycle. In this review, we will examine the ability of zebrafish and newts to respond to cardiac damage with de novo cardiogenesis, whereas rodents and humans respond with a marked fibrogenic response and virtually no cardiomyocyte regeneration. Concerted strategies are needed to overcome this evolutionarily imposed barrier and optimize cardiac regeneration in mammals.
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Anfibios/fisiología , Corazón/fisiología , Regeneración/fisiología , Pez Cebra/fisiología , Animales , Ciclo Celular/fisiología , Fibroblastos/fisiología , Peces , Humanos , Mamíferos , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Cell-mediated cardiac regeneration remains a challenge as a therapeutic option in heart failure, but modest success using experimental models suggests that a better understanding of normal histogenesis will be needed to make progress towards cardiac regeneration. Recent studies of the heart show that the interstitium informs organogenesis and responsiveness to pathological stimuli through continuous bidirectional cross-talk between cardiomyocytes and non-cardiac cells. Here, we introduce the concept of the "cardiovascular unit" (CVU) as a building block of the heart, which includes cardiomyocytes and adjacent capillaries and fibroblasts. We discuss how the CVU might be used as a tool for re-interpreting degenerative changes of the myocardium during aging and hypertrophy, and might represent the hallmark for successful cell therapy strategies in cardiac regeneration.
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Comunicación Celular/fisiología , Corazón/fisiología , Miocardio , Regeneración , Envejecimiento , Cardiomegalia/metabolismo , Cardiomegalia/patología , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Corazón/embriología , Insuficiencia Cardíaca/terapia , Humanos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Neovascularización Fisiológica , Organogénesis , Medicina Regenerativa , Transducción de Señal , Trasplante de Células Madre/tendenciasRESUMEN
The potential for in vitro colonization of decellularized valves by human bone marrow mesenchymal stem cells (hBM-MSCs) towards the anisotropic layers ventricularis and fibrosa and in homo- vs. heterotypic cell-ECM interactions has never been investigated. hBM-MSCs were expanded and characterized by immunofluorescence and FACS analysis. Porcine and human pulmonary valve leaflets (p- and hPVLs, respectively) underwent decellularization with Triton X100-sodium cholate treatment (TRICOL), followed by nuclear fragment removal. hBM-MSCs (2x10(6) cells/cm(2)) were seeded onto fibrosa (FS) or ventricularis (VS) of decellularized PVLs, precoated with FBS and fibronectin, and statically cultured for 30 days. Bioengineered PVLs revealed no histopathological features but a reconstructed endothelium lining and the presence of fibroblasts, myofibroblasts and SMCs, as in the corresponding native leaflet. The two valve layers behaved differently as regards hBM-MSC repopulation potential, however, with a higher degree of 3D spreading and differentiation in VS than in FS samples, and with enhanced cell survival and colonization effects in the homotypic ventricularis matrix, suggesting that hBM-MSC phenotypic conversion is strongly influenced in vitro by the anisotropic valve microstructure and species-specific matching between extracellular matrix and donor cells. These findings are of particular relevance to in vivo future applications of valve tissue engineering.
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Células de la Médula Ósea/citología , Matriz Extracelular/química , Prótesis Valvulares Cardíacas , Válvulas Cardíacas/ultraestructura , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Materiales Biomiméticos , Células de la Médula Ósea/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Válvulas Cardíacas/química , Humanos , Células Madre Mesenquimatosas/fisiología , Fenotipo , Diseño de Prótesis , Porcinos , Ingeniería de Tejidos/instrumentación , Adulto JovenRESUMEN
In the last 10 years an increasing interest has been devoted to the study of endothelial progenitor cells (EPCs), a subtype of immature cells involved in endothelial repair and neoangiogenesis. EPCs have been discovered as a novel integrated part of the cardiovascular system, which plays a comprehensive role in tissue homeostasis. Consistently, alterations and/or reduction of the circulating EPC pool have been associated with different manifestations of cardiovascular disorders and atherosclerosis. This is why, the extent of the EPC pool is now considered a mirror of vascular health, while EPC reduction has become a surrogate biomarker of cardiovascular risk and of the ongoing vascular damage. Unfortunately, the methods used to study EPCs still lack standardization, and this is significantly decelerating progress in the field. In this review, we focus on some aspects related to the two methods used to assess circulating EPCs: flow cytometry and cell culture. We uncover the many traps hidden in the choice of the right protocol, and suggest the best solutions on the basis of evidence and background theories.
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Antígenos CD/clasificación , Células Endoteliales/citología , Células Madre/citología , Aterosclerosis/fisiopatología , Biomarcadores/sangre , Técnicas de Cultivo de Célula , Células Endoteliales/fisiología , Citometría de Flujo/métodos , Humanos , Neovascularización Fisiológica/fisiología , Juego de Reactivos para Diagnóstico , Células Madre/fisiologíaRESUMEN
Atherosclerotic diseases are responsible for a significant part of morbidity and mortality in western countries. According to the classical views, atherosclerotic lesions develop as the result of an inflammatory process initiated by endothelial damage. The discovery that bone marrow-derived cells participate in endothelial repair and new vessel growth has changed the pathogenetic models of cardiovascular disease. These cells, termed endothelial progenitor cells (EPCs), represent the endogenous endothelial regenerative capacity and the ability to form new collateral vessels. In this review we describe how quantitative and qualitative alterations of EPCs have a significant role in virtually all stages of the atherosclerotic process and in the clinical manifestations of the diseases: starting from the impact of risk factors on EPCs, through the mechanisms that link EPC reduction/dysfunction to plaque formation, and finally to the clinical syndromes. An attempt to diverge our attention from the vessel wall to the bloodstream reveals a central role of EPCs in atherogenesis.
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Aterosclerosis/epidemiología , Aterosclerosis/patología , Células Endoteliales/patología , Endotelio Vascular/patología , Células Madre/patología , Humanos , Factores de RiesgoRESUMEN
PURPOSE: Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. MATERIALS AND METHODS: The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. RESULTS: At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. CONCLUSIONS: In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism.
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Líquido Amniótico/citología , Células de la Médula Ósea , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Músculo Liso/citología , Músculo Liso/patología , Trasplante de Células Madre , Vejiga Urinaria/citología , Animales , Femenino , Hipertrofia/prevención & control , Ratas , Ratas DesnudasRESUMEN
OBJECTIVE: To study the adaptive potential of the right ventricular myocardium after 30 days of mechanical-induced overload in rats from two different age groups. MATERIALS AND METHODS: We banded the pulmonary trunk, so as to increase the systolic work load of the right ventricle, in 19 adult Sprague-Dawley rats at the age of 10 weeks, and 16 weanlings when they were 3 weeks-old, using 10 adults and 10 weanlings as controls. We analysed the functional adaptation and structural changes of the right ventricular myocardium, blood vessels and interstitial tissue after 30 days of increased afterload. RESULTS: The increased workload induced an increase of the right ventricular weight and free wall thickness in animals from both age groups when compared to controls. These changes were mostly related to cardiomyocytic hypertrophy, as confirmed by the expression of myocardial hypertrophic markers, without any apparent increase of their number, a "reactive" fibrosis especially evident in the adult rats, with p-value less than 0.0001, and a more extensive neocapillary network in the weanlings compared to the adults aubsequent to banding, the p-value being less than 0.0001. CONCLUSION: In response to right ventricular afterload, weanlings showed a higher adaptive capillary growth, which hampered the development of fibrosis as seen in the adult rats. Age seems to be a risk factor for adverse structural-functional changes of right ventricle subjected to increased workload.