RESUMEN
Vasospasm is one of the important causes of morbidity in free flap and replantation surgery. In secondary Raynaud's phenomenon, nearly half of the patients experience digital ulceration, pain and loss of function at least once in their lifetime. The aim of this study is to investigate the vasodilation effect of ethanol-mediated chemical denervation on peripheral vessels by topical administration. In this study, 27 Wistar albino male rats weighing 250-300 grams were used. The rats were randomly divided into three groups: saline (group S, n = 8), lidocaine (group L, n = 9) and 96% ethanol (group E, n = 9). According to group, 0.1 mL saline, 0.1 mL lidocaine and 0.1 mL ethanol were applied around the rat femoral neurovascular bundle. After the application, on the 0th day and 3th weeks, femoral artery and vein diameters were measured. After 3. weeks, histopathological samples from femoral artery, vein and nerve were evaluated. On the 0th day, the mean diameter of the femoral artery and vein was similar in group E and L and higher than group S. After three weeks, the vasodilatation effect of ethanol was increased in group E. In Group L and S, the vasodilatation effect was lost. Histopathological examination showed that ethanol significantly caused perivascular inflammation and nerve degeneration compared to other agents and did not cause endothelial damage. Vasodilatation obtained by ethanol is a rapid onset and long-lasting effect. It is also inexpensive and effective for peripheral vasodilatation.
Asunto(s)
Etanol/farmacología , Arteria Femoral/efectos de los fármacos , Vena Femoral/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Administración Tópica , Animales , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Etanol/administración & dosificación , Arteria Femoral/citología , Vena Femoral/citología , Lidocaína/administración & dosificación , Masculino , Distribución Aleatoria , Ratas , Ratas Wistar , Solución Salina/administración & dosificaciónRESUMEN
Exercise is a treatment option in peripheral artery disease (PAD) patients to improve their clinical trajectory, at least in part induced by collateral growth. The ligation of the femoral artery (FAL) in mice is an established model to induce arteriogenesis. We intended to develop an animal model to stimulate collateral growth in mice through exercise. The training intensity assessment consisted of comparing two different training regimens in C57BL/6 mice, a treadmill implementing forced exercise and a free-to-access voluntary running wheel. The mice in the latter group covered a much greater distance than the former pre- and postoperatively. C57BL/6 mice and hypercholesterolemic ApoE-deficient (ApoE-/-) mice were subjected to FAL and had either access to a running wheel or were kept in motion-restricting cages (control) and hind limb perfusion was measured pre- and postoperatively at various times. Perfusion recovery in C57BL/6 mice was similar between the groups. In contrast, ApoE-/- mice showed significant differences between training and control 7 d postoperatively with a significant increase in pericollateral macrophages while the collateral diameter did not differ between training and control groups 21 d after surgery. ApoE-/- mice with running wheel training is a suitable model to simulate exercise induced collateral growth in PAD. This experimental set-up may provide a model for investigating molecular training effects.
Asunto(s)
Modelos Animales de Enfermedad , Neovascularización Fisiológica , Enfermedad Arterial Periférica/etiología , Condicionamiento Físico Animal , Animales , Biomarcadores , Dieta Alta en Grasa , Arteria Femoral/citología , Arteria Femoral/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/irrigación sanguínea , Enfermedad Arterial Periférica/metabolismo , Enfermedad Arterial Periférica/patología , Flujo Sanguíneo RegionalRESUMEN
Plasma membrane Na+/Ca2+ exchanger-1 (NCX1) helps regulate the cytosolic Ca2+ concentration ([Ca2+]CYT) in arterial myocytes. NCX1 mediates both Ca2+ entry and exit and tends to promote net Ca2+ entry in partially constricted arteries. Mean blood pressure (telemetry) is elevated by ≈10 mmHg in transgenic (TG) mice that overexpress NCX1 specifically in smooth muscle. We tested the hypothesis that NCX1 overexpression mediates Ca2+ gain and elevated [Ca2+]CYT in exposed femoral arteries that also express the Ca2+ biosensor exogenous myosin light chain kinase. [Ca2+]CYT and the NCX1-dependent (SEA0400-sensitive) component, ≈15% of total basal constriction in controls, were increased in TG arteries, but constrictions to phenylephrine and ANG II were comparable in TG and control arteries. Normalized phenylephrine dose-response curves and constriction to 30 and 300 ng/kg iv ANG II were virtually identical in control and TG arteries. ANG II-evoked constrictions, superimposed on elevated basal tone, accounted for the larger blood pressure responses to ANG II in TG arteries. TG and control mouse arteries fit the same pCa-constriction relationship over a wide range of pCa (≈125-500 nM). Vasodilation to acetylcholine, normalized to passive diameter, was also comparable in TG and control arteries, implying normal endothelial function. TG artery Na+ nitroprusside (nitric oxide donor)-induced dilations were, however, shifted to lower Na+ nitroprusside concentrations, indicating that TG myocyte vasodilator mechanisms were augmented. Maximum arterial dilation was comparable in TG and control mice, although passive diameter was ≈6-7% smaller in TG mice. The changes in TG arteries were apparently largely functional rather than structural, despite the congenital hypertension. NEW & NOTEWORTHY Smooth muscle Na+/Ca2+ exchanger-1 transgene overexpression (TG mice) increases femoral artery basal cytosolic Ca2+ concentration ([Ca2+]CYT) and tone in vivo and raises blood pressure. Arterial constriction to phenylephrine and angiotensin II are normal but superimposed on the augmented basal [Ca2+]CYT and tone (constriction) in TG mouse arteries. Similar effects in resistance arteries would explain the elevated blood pressure. Acetylcholine-induced vasodilation is unimpaired, implying a normal endothelium, but TG arteries are hypersensitive to sodium nitroprusside.
Asunto(s)
Calcio/metabolismo , Arteria Femoral/metabolismo , Músculo Liso Vascular/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Presión Sanguínea , Arteria Femoral/citología , Arteria Femoral/fisiología , Ratones , Ratones Endogámicos C57BL , Tono Muscular , Músculo Liso Vascular/fisiología , Óxido Nítrico/metabolismo , Intercambiador de Sodio-Calcio/genética , VasodilataciónRESUMEN
Pacinian corpuscles are vibration-sensing mechanoreceptors that are densely distributed in the dermis of the human hand. Although they are also known to occur in various other regions/structures throughout the human body, including the adventitia of large vessels, their precise distribution and function in arteries remain unclear. In the present study, we identified Pacini-like lamellar corpuscles (LCs) adjacent to the femoral artery, and investigated their distribution with respect to that structure via a histological analysis. We identified nine LCs that were localized in the connective tissue surrounding the femoral artery and vein. We showed that although their distribution was heterogeneous, they were predominantly concentrated on the dorsal side of the femoral artery. Immunohistochemical analyses revealed that the identified femoral artery LCs exhibited features characteristic of typical LCs located in the dermis of the index finger. Thus, the results of the present study contribute to an improved understanding of the function of femoral artery LCs. Anat Rec, 301:1809-1814, 2018. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Arteria Femoral/citología , Corpúsculos de Pacini/citología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , MasculinoRESUMEN
Vascularization of engineered tissues remains one of the key problems. Here, we described a novel approach to promote vascularization of engineered tissues using fibronectin (FN) incorporated gold nanoparticles (AuNP) coated onto catheters with mesenchymal stem cells (MSCs) for tissue engineering. We found that the FN-AuNP composite with 43.5 ppm of AuNP exhibited better biomechanical properties and thermal stability than pure FN. FN-AuNP composites promoted MSC proliferation and increased the biocompatibility. Mechanistically, vascular endothelial growth factor (VEGF) promoted MSC migration on FN-AuNP through the endothelial oxide synthase (eNOS)/metalloproteinase (MMP) signaling pathway. Vascular femoral artery tissues isolated from the implanted FN-AuNP-coated catheters with MSCs expressed substantial CD31 and alpha-smooth muscle actin (α-SMA), displayed higher antithrombotic activity, as well as better endothelialization ability than those coated with all other materials. These data suggested that the implantation of FN-AuNP-coated catheter with MSCs could be a novel strategy for vascular biomaterials applications.
Asunto(s)
Arteria Femoral/citología , Fibronectinas/química , Oro/química , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Nanopartículas del Metal/administración & dosificación , Ingeniería de Tejidos/métodos , Catéteres , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Arteria Femoral/fisiología , Humanos , Ensayo de Materiales , Nanopartículas del Metal/química , Regeneración , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de HeridasRESUMEN
The important factors that affect the arterial wall compliance are the tissue properties of the arterial wall, the in vivo pulsatile pressure, and the prestressed condition of the artery. It is necessary to obtain the load-free geometry for determining the physiological level of prestress in the arterial wall. The previously developed optimization-based inverse algorithm was improved to obtain the load-free geometry and the wall prestress of an idealized tapered femoral artery of a dog under varying arterial wall properties. The compliance of the artery was also evaluated over a range of systemic pressures (72.5-140.7 mmHg), associated blood flows, and artery wall properties using the prestressed arterial geometry. The results showed that the computed load-free outer diameter at the inlet of the tapered artery was 6.7%, 9.0%, and 12% smaller than the corresponding in vivo diameter for the 25% softer, baseline, and 25% stiffer arterial wall properties, respectively. In contrast, the variations in the prestressed geometry and circumferential wall prestress were less than 2% for variable arterial wall properties. The computed compliance at the inlet of the prestressed artery for the baseline arterial wall property was 0.34%, 0.19%, and 0.13% diameter change/mmHg for time-averaged pressures of 72.5, 104.1, and 140.7 mmHg, respectively. However, the variation in compliance due to the change in arterial wall property was less than 6%. The load-free and prestressed geometries of the idealized tapered femoral artery were accurately (error within 1.2% of the in vivo geometry) computed under variable arterial wall properties using the modified inverse algorithm. Based on the blood-arterial wall interaction results, the arterial wall compliance was influenced significantly by the change in average pressure. In contrast, the change in arterial wall property did not influence the arterial wall compliance.
Asunto(s)
Algoritmos , Arteria Femoral/fisiología , Hemodinámica , Estrés Mecánico , Arteria Femoral/citología , Análisis de Elementos Finitos , Modelos Cardiovasculares , Presión , Flujo PulsátilRESUMEN
AIMS: Our aim was to evaluate arterial responses to paclitaxel and a novel fluorocopolymer-coated nitinol low-dose paclitaxel-eluting stent (FP-PES). METHODS AND RESULTS: Human smooth muscle cell (SMC) migration was assessed after exposure to paclitaxel in vitro. For pharmacokinetics and vascular response, FP-PES or bare metal stents (BMS) were implanted in porcine iliofemoral arteries. Paclitaxel significantly inhibited human coronary and femoral artery SMC migration at doses as low as 1 pM. Inhibition was significantly greater for femoral compared with coronary artery SMCs from 1 pM to 1 µM. Pharmacokinetics showed consistent paclitaxel release from FP-PES over the study duration. The peak arterial wall paclitaxel level was 3.7 ng/mg at 10 days, with levels decreasing to 50% of peak at 60 days and 10% at 180 days. Paclitaxel was not detected in blood or remote organs. Arteriogram and histomorphometry analyses showed FP-PES significantly inhibits neointimal proliferation versus BMS at 30 and 90 days. Re-endothelialisation scores were not different between groups. CONCLUSIONS: Paclitaxel affected femoral artery SMC migration at lower concentrations and to a greater degree than it did coronary artery SMCs. The novel FP-PES used in this preclinical study demonstrated a vascular healing response similar to BMS, while significantly inhibiting neointimal formation up to 90 days.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Stents Liberadores de Fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Paclitaxel/administración & dosificación , Aleaciones , Animales , Antineoplásicos Fitogénicos/farmacocinética , Movimiento Celular/efectos de los fármacos , Vasos Coronarios/citología , Arteria Femoral/citología , Humanos , Modelos Animales , Neointima/prevención & control , Paclitaxel/farmacocinética , Polímeros , PorcinosRESUMEN
Hypoxia triggers pulmonary vasoconstriction, however induces relaxation of systemic arteries such as femoral arteries. Mitochondria are functionally and structurally heterogeneous between different cell types. The aim of this study was to reveal whether mitochondrial heterogeneity controls the distinct responses of pulmonary versus systemic artery smooth muscle cells to hypoxia. Intact mitochondria were transplanted into Sprague-Dawley rat pulmonary artery smooth muscle cells in culture and pulmonary arteries in vitro. Mitochondria retained functional after transplantation. The cross transplantation of mitochondria between pulmonary and femoral artery smooth muscle cells reversed acute hypoxia-induced alterations in cell membrane potential, [Ca2+]i signaling in smooth muscle cells and constriction or relaxation of arteries. Furthermore, the high or low amount of reactive oxygen species generation from mitochondria and their divergent (dis-)abilities in activating extracellular Ca2+-sensing receptor in smooth muscle cells were found to cause cell membrane potential depolarization, [Ca2+]i elevation and constriction of pulmonary arteries versus cell membrane potential hyperpolarization, [Ca2+]i decline and relaxation of femoral arteries in response to hypoxia, respectively. Our findings suggest that mitochondria necessarily determine the behaviors of vascular smooth muscle cells in response to hypoxia.
Asunto(s)
Mitocondrias/trasplante , Miocitos del Músculo Liso/fisiología , Arteria Pulmonar/fisiología , Vasoconstricción , Animales , Calcio/metabolismo , Hipoxia de la Célula , Células Cultivadas , Arteria Femoral/citología , Arteria Femoral/metabolismo , Arteria Femoral/fisiología , Hipoxia , Potenciales de la Membrana/fisiología , Microscopía Electrónica , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Evidence from human and animal studies has demonstrated elevated levels of the cysteine protease cathepsin S (CatS) in hypoxic atherosclerotic lesions. We hypothesized that silencing of CatS gene would suppress ischemia-induced angiogenic action. METHODS AND RESULTS: Left femoral artery ligation-induced ischemia in mice showed the increased expression and activity of CatS in the ischemic muscle. The CatS-deficiency (CatS(-/-)) mice showed impaired functional recovery following hindlimb ischemia and reduced levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), phospho-Akt (p-Akt), p-endothelial nitric oxide synthase, p-extracellular signal-regulated kinase1/2 (Erk1/2), p-p38 mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) proteins, as well as reduced levels of matrix metalloproteinase-9 and macrophage infiltration in the ischemic muscles. In vitro, CatS silencing reduced the levels of these targeted essential molecules for angiogenesis and vasculogenesis. Together, the results indicated that the effects of CatS knockdown led to defective endothelial cell invasion, proliferation, and tube formation. This notion was reinforced by the finding that CatS inhibition led to a decreased PPAR-γ level and VEGF/Erk1/2 signaling activation in response to ischemia. CatS(-/-) resulted in decreased circulating EPC-like CD31(+)/c-Kit(+) cells, accompanied by the reduction of the cellular levels of PPAR-γ, p-Akt, and VEGF induced by ischemic stress. Transplantation of bone-marrow-derived mononuclear cells from CatS(+/+) mice restored neovascularization in CatS(-/-) mice. CONCLUSIONS: CatS activity controls ischemia-induced neovascularization partially via the modulation of PPAR-γ and VEGF/Akt signaling activation.
Asunto(s)
Catepsinas/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigación sanguínea , Animales , Células Endoteliales/citología , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Arteria Femoral/citología , Arteria Femoral/enzimología , Arteria Femoral/metabolismo , Miembro Posterior/irrigación sanguínea , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Neovascularización Patológica/enzimología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Madre/citología , Células Madre/enzimología , Células Madre/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Recent evidence from humans and rats indicates that nitrite is a vasodilator under hypoxic conditions by reacting with metal-containing proteins to produce nitric oxide (NO). We tested the hypothesis that near-physiological concentrations of nitrite would produce vasodilation in a hypoxia- and concentration-dependent manner in the hind limb of sheep. Anesthetized sheep were instrumented to measure arterial blood pressure and femoral blood flows continuously in both hind limbs. Nitrite was infused into one femoral artery to raise the nitrite concentration in the femoral vein by 10 to 15-fold while the sheep breathed 50%, 14% or 12% oxygen in inspired air. In contrast to reports in humans and rats, the nitrite infusion had no measurable effect on mean femoral blood flows or vascular conductances, regardless of inspired O2 levels. In vitro experiments showed no significant difference in the release of NO from nitrite in sheep and human red blood cells. Further experiments demonstrated nitrite is converted to NO in rat artery homogenates faster than sheep arteries, and that this source of NO production is attenuated in the presence of a heme oxidizer. Finally, western blots indicate that concentrations of the heme-containing protein cytoglobin, but not myoglobin, are markedly lower in sheep arteries compared with rats. Overall, the results demonstrate that nitrite is not a physiological vasodilator in sheep. This is likely due to a lack of conversion of nitrite to NO within the vascular smooth muscle, perhaps due to deficient amounts of the heme-containing protein cytoglobin.
Asunto(s)
Músculo Liso Vascular/fisiología , Nitritos/sangre , Vasodilatación , Animales , Citoglobina , Femenino , Arteria Femoral/citología , Arteria Femoral/metabolismo , Arteria Femoral/fisiología , Globinas/metabolismo , Miembro Posterior/irrigación sanguínea , Hipoxia/sangre , Hipoxia/metabolismo , Hipoxia/fisiopatología , Masculino , Músculo Liso Vascular/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxígeno/sangre , Ratas , Flujo Sanguíneo Regional , OvinosRESUMEN
The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.
Asunto(s)
Prótesis Vascular , Arteria Femoral/citología , Arteria Femoral/fisiología , Arterias Mamarias/citología , Arterias Mamarias/fisiología , Ingeniería de Tejidos/métodos , Animales , Fenómenos Biomecánicos , Bovinos , Endotelio/fisiología , Femenino , Humanos , Perfusión , Andamios del TejidoRESUMEN
Physiological loads that act on the femoropopliteal artery, in combination with stenting, can lead to uncharacteristic deformations of the stented vessel. The overall goal of this study was to investigate the effect of stent length and stent location on the deformation characteristics of the superficial femoral artery (SFA) using an anatomically accurate, three-dimensional finite element model of the leg. For a range of different stent lengths and locations, the deformation characteristics (length change, curvature change, and axial twist) that result from physiological loading of the SFA along with the mechanical behavior of the vessel tissue are investigated. Results showed that stenting portions of the SFA leads to a change in global deformation characteristics of the vessel. Increased stress and strain values and altered deformation characteristics were observed in the various stented cases of this study, which are compared to previous results of an unstented vessel. The study concludes that shortening, twist and curvature characteristics of the stented vessel are dependent on stent length and stent location within the vessel.
Asunto(s)
Arteria Femoral/anatomía & histología , Arteria Femoral/fisiología , Análisis de Elementos Finitos , Fenómenos Mecánicos , Arteria Poplítea/anatomía & histología , Arteria Poplítea/fisiología , Stents , Fenómenos Biomecánicos , Arteria Femoral/citología , Arteria Poplítea/citología , Estrés MecánicoRESUMEN
BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Fosfatasa 1/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Vasoconstricción/fisiología , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Señalización del Calcio/fisiología , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Arteria Femoral/citología , Arteria Femoral/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Arterias Mamarias/citología , Arterias Mamarias/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Fenotipo , Proteína Fosfatasa 1/genética , Proteínas/genética , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismoRESUMEN
Vascular calcification is associated with significant cardiovascular morbidity and mortality, and has been demonstrated as an actively regulated process resembling bone formation. Oxidized low density lipoprotein (Ox-LDL) has been identified as a regulatory factor involved in calcification of vascular smooth muscle cells (VSMCs). Additionally, over-expression of recombinant human neutral sphingomyelinase (N-SMase) has been shown to stimulate VSMC apoptosis, which plays an important role in the progression of vascular calcification. The aim of this study is to investigate whether ceramide regulates Ox-LDL-induced calcification of VSMCs via activation of p38 mitogen-activated protein kinase (MAPK) pathway. Ox-LDL increased the activity of N-SMase and the level of ceramide in cultured VSMCs. Calcification and the osteogenic transcription factor, Msx2 mRNA expression were reduced by N-SMase inhibitor, GW4869 in the presence of Ox-LDL. Usage of GW4869 inhibited Ox-LDL-induced apoptosis in VSMCs, an effect which was reversed by C2-ceramide. Additionally, C2-ceramide treatment accelerated VSMC calcification, with a concomitant increase in ALP activity. Furthermore, C2-ceramide treatment enhanced Ox-LDL-induced VSMC calcification. Addition of caspase inhibitor, ZVAD-fmk attenuated Ox-LDL-induced calcification. Both Ox-LDL and C2-ceramide treatment increased the phosphorylation of p38 MAPK. Inhibition of p38 MAPK by SB203580 attenuated Ox-LDL-induced calcification of VSMCs. These data suggest that Ox-LDL activates N-SMase-ceramide signaling pathway, and stimulates phosphorylation of p38 MAPK, leading to apoptosis in VSMCs, which initiates VSMC calcification.
Asunto(s)
Ceramidas/metabolismo , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal/fisiología , Calcificación Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Compuestos de Bencilideno/farmacología , Células Cultivadas , Ceramidas/farmacología , Arteria Femoral/citología , Arteria Femoral/efectos de los fármacos , Arteria Femoral/metabolismo , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Fosforilación , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias , Células Endoteliales , Endotelio Vascular , Arteria Femoral , Animales , Antígenos de Diferenciación/biosíntesis , Línea Celular , Citocinas/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Arteria Femoral/citología , Arteria Femoral/metabolismo , Humanos , PapioRESUMEN
Natural extracellular matrix (ECM) deposited in situ by cultured endothelial cells (ECs) has been proven effective in accelerating endothelialization of titanium (Ti) cardiovascular implants (CVIs) in our previous studies. In this study, the ECM deposited by smooth muscle cells (SMCs) was used in comparison to investigate the effects of tissue specificity of the ECM on the ability to accelerate endothelialization of CVIs. The results demonstrated that the ECM deposited by ECs and SMCs (EC-ECM, SMC-ECM, respectively) differed considerably in components and fibril morphology. Surface modification of Ti CVIs with both types of natural ECM was effective in improving their in vitro hemocompatibility and cytocompatibility simultaneously. However, the endothelialization of ECM-modified Ti CVIs in a canine model demonstrated a high tissue specificity of the ECM. Although the ECM deposited by SMCs (SMC-ECM) induced fewer platelet adhesion and sustained better growth and viability of ECs in vitro, its performance in accelerating in vivo endothelialization of Ti CVIs was extremely poor. In contrast, the ECM deposited by ECs (EC-ECM) led to complete endothelium formation in vivo.
Asunto(s)
Prótesis Vascular , Endotelio Vascular/citología , Endotelio Vascular/crecimiento & desarrollo , Matriz Extracelular/metabolismo , Arteria Femoral/citología , Miocitos del Músculo Liso/citología , Andamios del Tejido , Animales , Células Cultivadas , Perros , Arteria Femoral/metabolismo , Arteria Femoral/cirugía , Miocitos del Músculo Liso/metabolismo , Especificidad de Órganos/fisiologíaRESUMEN
The objective of this study was to design and test a protocol for the validation of banking methodologies for arterial allografts. A series of in vitro biomechanical and biological assessments were derived, and applied to paired fresh and banked femoral arteries. The ultimate tensile stress and strain, suture pullout stress and strain, expansion/rupture under hydrostatic pressure, histological structure and biocompatibility properties of disinfected and cryopreserved femoral arteries were compared to those of fresh controls. No significant differences were detected in any of the test criteria. This validation protocol provides an effective means of testing and validating banking protocols for arterial allografts.
Asunto(s)
Aloinjertos/fisiología , Arteria Femoral/trasplante , Bancos de Tejidos/normas , Conservación de Tejido/métodos , Adulto , Fenómenos Biomecánicos , Línea Celular , Criopreservación , Femenino , Arteria Femoral/citología , Arteria Femoral/fisiología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Presión , Estándares de Referencia , Reproducibilidad de los Resultados , Suturas , Resistencia a la Tracción , Adulto JovenRESUMEN
The purpose of the present study was to compare the differences between the role of volume-regulated Clâ» channels (VRCCs) in veins and arteries. We used the whole cell patch clamp and fluorescence imaging techniques to evaluate swelling-induced Clâ» current (I(Cl,vol)) and changes in the intracellular concentrations of Clâ» ([Clâ»](i)) induced by hypotonic solutions in rat femoral artery cells (FASMCs) and vein smooth muscle cells (FVSMCs). I(Cl,vol) and [Clâ»](i) decline induced by hypotonic solution were more prominent in FASMCs than in FVSMCs. I(Cl,vol) and the alterations in [Clâ»](i) were gradually increased as the number of cell passages increased. However, the regulatory function of tyrosine protein phosphorylation in volume-regulated chloride movement is prominent in veins. The expression of ClC-3 was higher in FASMCs than in FVSMCs. VRCC activity is more pronounced in rat femoral arteries than in veins. VRCC activity and tyrosine protein phosphorylation regulative function increase gradually as vascular cells switch from contractile to proliferative phenotypes.
Asunto(s)
Canales de Cloruro/metabolismo , Arteria Femoral/metabolismo , Vena Femoral/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatación , Animales , Transporte Biológico , Línea Celular Transformada , Proliferación Celular , Células Cultivadas , Cloruros/metabolismo , Arteria Femoral/citología , Vena Femoral/citología , Masculino , Potenciales de la Membrana , Músculo Liso Vascular/citología , Concentración Osmolar , Técnicas de Placa-Clamp , Fosforilación , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Combretastatin-A4-phosphate (CA4P/CA4), an anti-cancer drug, induces tumour hypoxia by destabilizing the cytoskeleton in tumour endothelial cells. Hypertensive side effects have been observed. We hypothesized that CA4P/CA4 lead to endothelial dysfunction followed by increased vasoconstriction. Mesenteric small arteries and femoral arteries isolated from male Wistar rats were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS). Immunoblotting of endothelial nitric oxide synthase (eNOS) was performed on human umbilical vein endothelial cells (HUVECs). CA4P failed per se to change vascular tone. In femoral arteries, endothelial cell removal, l-nitro-arginine (l-NNA, an inhibitor of eNOS) and CA4P enhanced phenylephrine-induced vasoconstriction, while in mesenteric arteries only l-NNA leftward shifted concentration-response curves for phenylephrine. CA4P enhanced vasoconstriction induced by low frequency (0.5-4Hz) EFS in femoral arteries, but not in mesenteric arteries. Neurogenic contractions were inhibited by prazosin, an α(1)-adrenoceptor antagonist. In mesenteric arteries, CA4P and l-NNA inhibited vasorelaxation induced by vanadate, a tyrosine phosphatase inhibitor. CA4P did not affect acetylcholine-induced relaxation. In HUVECs, CA4P increased phosphorylation at eNOS-Thr(495), a negative regulatory site, while the positive phosphorylation site eNOS-Ser(1177) was not affected. CA4 neither influenced the actions of phenylephrine, vanadate nor acetylcholine in femoral and mesenteric arteries. In conclusion, our findings suggest that CA4P, but not CA4, enhances sympathetic adrenergic vasoconstriction probably by increasing eNOS-Thr(495) phosphorylation, in a tissue selective manner. These findings encourage further investigation to show that the hypertension and regional organ ischemia induced by CA4P can be avoided by concomitant treatment with an α(1)-adrenoceptor antagonist.
Asunto(s)
Arteria Femoral/efectos de los fármacos , Arteria Femoral/fisiología , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Estilbenos/efectos adversos , Sistema Nervioso Simpático/fisiología , Vasoconstricción/efectos de los fármacos , Acetilcolina/farmacología , Animales , Antineoplásicos/efectos adversos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Arteria Femoral/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Técnicas In Vitro , Masculino , Arterias Mesentéricas/citología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenilefrina/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Sistema Nervioso Simpático/efectos de los fármacos , Vanadatos/farmacologíaRESUMEN
We report expression of Pax3, an important regulator of skeletal muscle stem cell behaviour, in the brachial and femoral arteries of adult mice. In these contractile arteries of the limb, but not in the elastic arteries of the trunk, bands of GFP-positive cells were observed in Pax3(GFP/+) mice. Histological and biochemical examination of the vessels, together with clonal analysis after purification of Pax3-GFP-positive cells by flow cytometry, established their vascular smooth muscle identity. These blood-vessel-derived cells do not respond to inducers of other mesodermal cell types, such as bone, however, they can contribute to muscle fibre formation when co-cultured with skeletal muscle cells. This myogenic conversion depends on the expression of Pax3, but is rare and non-cell autonomous as it requires cell fusion. Myocardin, which promotes acquisition of a mature smooth muscle phenotype in these Pax3-GFP-positive cells, antagonises their potential for skeletal muscle differentiation. Genetic manipulation shows that myocardin is, however, positively regulated by Pax3, unlike genes for other myocardin-related factors, MRTFA, MRTFB or SRF. Expression of Pax3 overlaps with that reported for Msx2, which is required for smooth muscle differentiation of blood vessel-derived multipotent mesoangioblasts. These observations are discussed with respect to the origin and function of Pax3-expressing cells in blood vessels, and more general questions of cell fate determination and adult cell plasticity and reprogramming.