RESUMEN
Aortic valve interstitial cells are responsible for maintaining the valve in response to their local mechanical environment. However, the complex organization of the extracellular matrix means cell strains cannot be directly derived from gross strains, and knowledge of tissue structure-function correlations is fundamental towards understanding mechanotransduction. This study investigates strain transfer through the valve, hypothesizing that organization of the valve matrix leads to non-homogenous local strains. Radial and circumferential samples were cut from aortic valve leaflets and subjected to quasi-static mechanical characterization. Further samples were imaged using confocal microscopy, to determine local strains in the matrix. Mechanical data demonstrated that the valve was significantly stronger and stiffer when loaded circumferentially, comparable with previous studies. Micromechanical studies demonstrated that strain transfer through the matrix is anisotropic and indirect, with local strains consistently smaller than applied strains in both orientations. Under radial loading, strains were transferred linearly to cells. However, under circumferential loading, strains were only one-third of applied values, with a less direct relationship between applied and local strains. This may result from matrix reorganization, and be important for preventing cellular damage during normal valve function. These findings should be taken into account when investigating interstitial cell behaviours, such as cell metabolism and mechanotransduction.
Asunto(s)
Válvula Aórtica/fisiología , Bioprótesis , Matriz Extracelular/fisiología , Prótesis Valvulares Cardíacas , Mecanotransducción Celular/fisiología , Animales , Fenómenos Biomecánicos , Elasticidad , Femenino , Microscopía Confocal , Estimulación Física , Resistencia al Corte , Estrés Mecánico , Porcinos , Resistencia a la Tracción/fisiologíaRESUMEN
Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.
Asunto(s)
Proteínas Contráctiles/metabolismo , Corazón/embriología , Proteínas de Microfilamentos/metabolismo , Animales , Endocardio/embriología , Endocardio/metabolismo , Filaminas , Humanos , Inmunohistoquímica , Mesodermo/embriología , Mesodermo/metabolismo , RatonesRESUMEN
Optimal function of the aortic root relies upon the ability of its component structures to move in a coordinated fashion. Some of the cells that make up the structures of the aortic root have been shown to contain nerves, receptors, and contractile elements. The ability to contract or relax may contribute to the successful function of the valve by allowing it to move in a coordinated manner in response to biological stimuli. It is known that cusp tissue receives primary, sensory, and autonomic nerves, suggesting a role for neuronal regulation of cusp function. In addition, cusp tissue has been shown to express a wide variety of receptors and to contract to a range of common vasoactive agents. The cells that constitute the valve have also shown secretory and proliferative responses. The biological signals that mediate the cross-talk between the different parts of the root have not been established. This review will examine the mechanisms that have been documented to be present and to assess their potential contribution in affecting aortic valve function.
Asunto(s)
Aorta Torácica/fisiología , Aorta Torácica/ultraestructura , Humanos , Función VentricularRESUMEN
Diaspirin cross-linked hemoglobin (DCL-Hb), when infused into animals, causes vasoconstriction thought to be caused by nitric oxide (NO) binding by the hemoglobin molecule. The purpose of this study was to ascertain whether DCL-Hb causes vasoconstriction in human saphenous vein taken from patients undergoing myocardial revascularization and whether NO scavenging is the mechanism. The direct effect of DCL-Hb on saphenous vein tone was tested by adding increasing concentrations (10(-8) to 10(-5)M) of the drug. In an additional series of experiments, the influence of DCL-Hb on the dilator response to endothelial dependent and independent vasodilators was tested. This was achieved by attempting either to reverse the effects of acetylcholine, sodium nitroprusside, or S-nitrosylglutathione with prior incubation with DCL-Hb or to inhibit the dilator response in vessels preconstricted with 10(-6)M norepinephrine. There was no effect of DCL-Hb alone on saphenous vein tone. DCL-Hb significantly reduced vasodilatation with all vasodilators (P < 0.05). After maximal relaxation with sodium nitroprusside and s-nitrosylglutathione, there was significant vasoconstriction with DCL-Hb at concentrations larger than 10(-6)M, (P < 0.05). The authors conclude that DCL-Hb does not constrict human saphenous vein but can affect vessel tone by reversal of the effect of endogenously or exogenously released NO.
Asunto(s)
Aspirina/análogos & derivados , Aspirina/farmacología , Sustitutos Sanguíneos/farmacología , Glutatión/análogos & derivados , Hemoglobinas/farmacología , Vena Safena/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Acetilcolina/farmacología , Glutatión/farmacología , Humanos , Técnicas In Vitro , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , S-Nitrosoglutatión , Vena Safena/fisiologíaRESUMEN
BACKGROUND AND AIM OF THE STUDY: The contraction of cusp tissue has been implicated to play a role in aortic valve function. The effect of the contractile agent 5-hydroxytryptamine (5-HT) on the competence of isolated aortic roots has been assessed, and the vasomotor properties of 5-HT on aortic root tissue examined. METHODS: Isolated porcine aortic roots were pressurized with Kreb's solution through the aortic arch. 5-HT was added in increasing concentrations (10(-7) to 10(-5) M) and the degree of leakage measured over time. In additional experiments, portions of sinotubular junction, sinus, annular and cusp tissue were set up in organ baths, placed under tension, and challenged with 5-HT (10(-9) to 10(-5) M). Viability of each valve structure was assessed by addition of KCl (90 mM). RESULTS: The rate of leakage from intact aortic roots increased when 10(-6) and 10(-5) M 5-HT was added. The maximum effect, observed at 10(-5) M 5-HT, was equal to an increase of 61.8+/-23.0% above control (p <0.05). The perfusion pressure at each concentration of 5-HT was unchanged. This response was inhibited by the 5-HT2A receptor antagonist ketanserin. Addition of KCl to isolated valve structures gave a mean contractile response of 0.8+/-0.1mN for cusp, 19+/-11.0 mN for annular, 29+/-8.0 mN for sinus, and 23+/-8.0 mN for sinotubular junction tissue (each n = 4). Only cusp tissue contracted when treated with 5-HT, with a maximum 105.5+/-17.2% (n = 4) of the response to KCl. The response to 5-HT was blocked by the 5-HT2A-receptor antagonist ketanserin at 10(-6) M (n = 4). None of the other aortic root structures responded to 5-HT. CONCLUSION: These results show that 5-HT influences the competence of isolated porcine aortic valves. This effect is contributed by contraction of the cusp tissue, and is mediated by 5-HT2A receptors. These effects may contribute to the association between valve dysfunction, 5-HT and certain appetite suppressants.
Asunto(s)
Insuficiencia de la Válvula Aórtica/fisiopatología , Válvula Aórtica/efectos de los fármacos , Válvula Aórtica/fisiopatología , Depuradores de Radicales Libres/farmacología , Serotonina/farmacología , Animales , Circulación Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Técnicas In Vitro , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , PorcinosRESUMEN
Occlusive accelerated atherosclerosis of coronary grafts is the predominant factor that limits longevity of heart transplant recipients. This form of vascular disease affects both the large epicardial and the smaller intramyocardial vessels, leading to characteristic clinical presentation that necessitates the use of sophisticated techniques for their accurate detection. Accelerated atherosclerosis after transplantation is a multifactorial disease with many events contributing to its progression. The initial vascular injury associated with ischemia-reperfusion appears to aggravate preexisting conditions in the donor vasculature in addition to activation of new immunological and nonimmunological mechanisms. Throughout these events, the endothelium remains a primary target of cell- and humoral-mediated injury. Changes in the vascular intima leads to alterations in vascular smooth muscle cell (VSMC) physiology, resulting in VSMC phenotypic modulation with the orchestration of a broad spectrum of growth and inflammatory reactions, which might be a healing response to vascular injury. Endogenous nitric oxide (NO) pathways regulate a multiplicity of cellular mechanisms that play a major role in determining the structure and function of the vessel wall during normal conditions and during remodeling associated with accelerated atherosclerosis. Recently identified signaling pathways, including mitogen-activated protein kinase, cGMP-dependent protein kinase, phosphatidylinositol 3-kinase, and transcriptional events in which nuclear factor kappa B and activator protein 1 take part, can be associated with NO modulation of cell cycle perturbations and phenotypic alteration of VSMC during accelerated atherosclerosis. This article reviews recent progress covering the aforementioned matters. We start by summarizing the clincal aspects and pathogenesis of accelerated atherosclerosis associated with transplantation, including clinical presentation and detection. This summary is followed by a discussion of the multiple factors of the disease process, including immunological and nonimmunolgical contributions. The next section focuses on cellular responses of the VSMCs relevant to lesion formation, with special emphasis on classical and recent paradigms of phenotypic modulation of these cells. To examine the influence of NO on VSMC phenotypic modulation and consequent lesion development, we briefly overview characteristics of NO production in the normal coronary vascular bed and the changes in endogenous NO release and activity during atherosclerosis. This overview is followed by a section covering molecular mechanisms whereby NO regulates a range of signaling pathways, transcriptional events underlying cell cycle perturbation, and phenotypic alteration of VSMC in accelerated atherosclerosis.
Asunto(s)
Enfermedad de la Arteria Coronaria/etiología , Trasplante de Corazón/efectos adversos , Músculo Liso Vascular/fisiopatología , Óxido Nítrico/fisiología , Animales , Enfermedad de la Arteria Coronaria/fisiopatología , Oclusión de Injerto Vascular/etiología , Oclusión de Injerto Vascular/fisiopatología , Humanos , FenotipoAsunto(s)
Angiotensina II/biosíntesis , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Vasos Sanguíneos/enzimología , Sistema Renina-Angiotensina/fisiología , Serina Endopeptidasas/metabolismo , Animales , Quimasas , Femenino , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
BACKGROUND AND AIM OF THE STUDY: The vasoactive agent 5-hydroxytryptamine (5-HT) has been implicated in valve disease due to possible trophic effects on valve interstitial cells (IC). The present study was aimed at characterizing the responses of cultured human heart valve IC to 5-HT in terms of intracellular calcium concentration ([Ca2+]i), mitogenesis and collagen synthesis. The effects of angiotensin II (Ang II) were also studied in parallel. METHODS: IC were obtained by collagenase digestion of valve leaflets isolated from transplant recipient hearts. Changes in [Ca2+]i were measured from fluorescence of the ratiometric calcium dye, fura 2. Mitogenic and collagen synthetic responses of valve IC were measured by 3H-thymidine incorporation (DNA synthesis) and 3H-proline incorporation assays respectively, in quiescent cells. RESULTS: Human valve IC responded to 5-HT and Ang II with mean maximal increases in [Ca2+]i of 249 +/- 47 nM and 397 +/- 159 nM, respectively. 5-HT stimulated DNA synthesis in quiescent IC, although to varying degrees among different isolations, with a maximum 43.4 +/- 20.1% increase by 10(-7) M 5-HT (p <0.05). Ang II did not stimulate IC DNA synthesis. Valve IC also responded to 5-HT with a maximum increase in collagen synthesis of 15.7 +/- 2.0% by 10(-6) M 5-HT (p <0.05). Ang II provoked a more powerful collagen synthesis response (maximum 50.5 +/- 15.1% increase by 10(-5) M Ang II; p <0.05). CONCLUSION: We have shown that 5-HT and Ang II promote the prolonged processes of growth and collagen synthesis in cultured human valve IC. Thus, these vasoactive agents may play a role in the development of heart valve disease.
Asunto(s)
Válvulas Cardíacas/efectos de los fármacos , Serotonina/farmacología , Angiotensina II/farmacología , Calcio/metabolismo , Células Cultivadas , Colágeno/biosíntesis , ADN/biosíntesis , Válvulas Cardíacas/citología , Humanos , Técnicas In Vitro , Vasoconstrictores/farmacologíaRESUMEN
1. The current study explored potential redox mechanisms of nitric oxide (NO)-induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells. 2. Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration-dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO. 3. Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO. 4. Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation. 5. Study of extracellula interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release. 6. Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells. 7. These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors.
Asunto(s)
ADN/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/farmacología , Acetilcisteína/farmacología , Animales , Ácido Ascórbico/farmacología , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Fase G1 , Glutatión/análogos & derivados , Glutatión/farmacología , Humanos , Hidroxiurea/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Oxidación-Reducción , Penicilamina/análogos & derivados , Penicilamina/farmacología , Ratas , Fase S , S-NitrosoglutatiónRESUMEN
BACKGROUND AND AIM OF THE STUDY: The mechanisms that regulate the function of the aortic valve are not fully understood. Cusp tissue has been shown to have contractile properties, but little is known as to which receptors mediate these effects. METHODS: We have examined, using isolated organ baths, the response of porcine aortic valve leaflets to a range of vasoactive agents including endothelin-1, noradrenaline, adrenaline, the thromboxane (TX) A2 mimetic U46619, 5-hydroxytryptamine (5-HT), histamine and angiotensin II. The viability of each individual leaflet was tested by the addition of 90 mM KCl. RESULTS: All agents tested, with the exception of angiotensin II, were capable of inducing concentration-dependent contractions of the valve leaflets. The responses to endothelin-1 and U46619 were significantly greater than those of all the other agents tested. Responses to endothelin-1 could be inhibited by 10(-5) M of the mixed ET(A/B) receptor antagonist bosantan. The response to both catecholamines was blocked by 10(-6) M yohimbine, but not by 3 x 10(-7) M prazosin, indicating the presence of alpha2-adrenoceptors. The response to histamine was mediated exclusively by H1-receptors, as judged by the antagonistic effect of 10(-6) M of the H1-receptor antagonist mepyramine. The response to 5-HT could be blocked by 10(-6) M of the 5-HT2A-receptor antagonist ketanserin, and that of U46619 by 10(-6) M of the TXA2-receptor antagonist SQ30741. CONCLUSION: These results demonstrate the range of receptor systems that can mediate contraction of aortic valve leaflets. Further studies are required to elucidate the role of these receptors in the physiology and pathophysiology of the aortic valve.
Asunto(s)
Válvula Aórtica/fisiología , Hemodinámica/fisiología , Contracción Miocárdica/fisiología , Receptores de Neurotransmisores/fisiología , Animales , Válvula Aórtica/efectos de los fármacos , Técnicas de Cultivo , Hemodinámica/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Receptores de Droga/efectos de los fármacos , Receptores de Droga/fisiología , Receptores de Neurotransmisores/efectos de los fármacos , Porcinos , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
The renin angiotensin system is implicated in the development of vein graft disease after coronary artery bypass surgery. Components of this system have been shown to play important roles in determining the short-term and long-term performance of coronary artery bypass grafts. Significant differences exist in the commonly used arterial and venous grafts in angiotensin converting enzyme activity and angiotensin responses. The existence of a dual enzyme pathway in angiotensin II formation has also been demonstrated. Such findings have implications for the use of AT1-receptor antagonists over enzyme inhibitors to improve graft performance and prevent the development of coronary artery bypass graft disease.
Asunto(s)
Puente de Arteria Coronaria , Enfermedad Coronaria/fisiopatología , Sistema Renina-Angiotensina , Angiotensina II/fisiología , Animales , Enfermedad Coronaria/cirugía , Endotelio Vascular/fisiología , Humanos , Contracción Muscular/fisiología , Peptidil-Dipeptidasa A/fisiología , Receptores de Angiotensina/fisiologíaRESUMEN
The aim of the present study was to investigate if the function of 5-hydroxytryptamine-1B/1D (5-HT1B/1D) receptors in human radial artery (RA) and internal thoracic artery (ITA) can be modified by thromboxane A2 (TXA2) released from the vessel wall in these two arteries that are commonly used in coronary artery bypass grafts. The 5-HT1B/1D agonist sumatriptan contracted the RA with a maximum response of 23.5+/-6.8 mN and a pD2 value of 6.6+/-0.1. The effect of sumatriptan was significantly reduced in the ITA with a maximum of 5.8+/-2.7 mN (P<.05) and a pD2 value of 6.4+/-0.2. The TXA2 receptor antagonist SQ30741 inhibited contractions to sumatriptan in the RA but not in the ITA. It is concluded that the effect mediated by 5-HT1B/1D is augmented by endogenous TXA2 in the RA.
Asunto(s)
Arterias Mamarias/efectos de los fármacos , Arteria Radial/efectos de los fármacos , Receptores de Serotonina/fisiología , Serotonina/farmacología , Tromboxano A2/fisiología , Anciano , Humanos , Técnicas In Vitro , Arterias Mamarias/fisiología , Persona de Mediana Edad , Arteria Radial/fisiología , Receptor de Serotonina 5-HT1B , Receptor de Serotonina 5-HT1D , Sumatriptán/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
BACKGROUND: The potential role of the local renin-angiotensin system to differentially affect radial artery and internal thoracic artery graft performance has not been examined. METHODS: Contractile responses to angiotensin I and II in the radial artery and the internal thoracic artery were examined in vitro. The expression function, and localization of angiotensin receptors, angiotensin converting enzyme, and chymase were studied in radial artery and internal thoracic artery segments. RESULTS: Angiotensin I and II contractions were significantly greater (p < 0.05) in the radial artery compared to the internal thoracic artery. In both arteries, angiotensin II responses were mediated via the AT1 receptor. Messenger RNA transcripts for angiotensin-converting enzyme and chymase were detected in both arteries. Angiotensin-converting enzyme was localized to luminal and vaso vasorum endothelial cells and smooth muscle cells in both vessels, while chymase was colocalized with mast cells in adventitial and medial layers. An angiotensin converting enzyme or a chymase inhibitor singularly had no effect on angiotensin I contractions, however, when combined, a marked inhibition of the angiotensin I response was observed in both vessels. CONCLUSIONS: Our results illustrate the complexities which exist within the local renin angiotensin system and suggest that clinical trials which may modulate the system are warranted.
Asunto(s)
Angiotensina II/fisiología , Arteria Radial/enzimología , Serina Endopeptidasas/fisiología , Arterias Torácicas/enzimología , Vasoconstricción/fisiología , Anciano , Quimasas , Técnicas de Cultivo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistema Renina-Angiotensina/fisiologíaRESUMEN
We investigated the influence of nitrovasodilators on DNA synthesis in cultured human aortic smooth muscle cells and explored the hypothesis that nitric oxide (NO) is directly involved in mediating the inhibitory effects of hydroxyurea on DNA synthesis. Both NO and hydroxyurea inhibited ongoing DNA synthesis and S phase progression in our cells. Exogenous deoxynucleosides partially reversed this inhibition, suggesting that ribonucleotide reductase is a primary target for both NO and hydroxyurea. Nitrovasodilators inhibited DNA synthesis by releasing NO, as detected by chemiluminescence and as shown by the reversal of DNA synthesis inhibition by NO scavengers. This inhibition appears to occur via a cGMP-independent mechanism. In contrast, hydroxyurea did not produce a detectable NO signal, and NO scavengers had no influence on its inhibition of DNA synthesis, suggesting that NO does not mediate the inhibitory action of hydroxyurea in our system. Furthermore, the action of nitrovasodilators and hydroxyurea on DNA synthesis differed according to redox sensitivity. The redox agents N-acetyl-L-cysteine and ascorbate reversed NO inhibition of DNA synthesis and had no effect on DNA synthesis inhibition caused by hydroxyurea.
Asunto(s)
ADN/biosíntesis , Hidroxiurea/metabolismo , Mercaptoetanol , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , S-Nitrosotioles , Antioxidantes/farmacología , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , ADN/antagonistas & inhibidores , Desoxiadenosinas/farmacología , Desoxiguanosina/farmacología , Humanos , Hidroxiurea/farmacología , Mediciones Luminiscentes , Músculo Liso Vascular/citología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacologíaRESUMEN
The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in inflammatory and proliferative vascular mechanisms. Activated NF-kappaB has been documented in human atherosclerotic lesions, and its activation in human vascular smooth muscle cells (SMC) by cytokines has been reported. However, intracellular mechanisms mediating NF-kappaB activation in human SMC are poorly understood. The aim of this study was to explore the potential role of reactive oxygen species and oxidant stress as signaling events in cytokine-induced NF-kappaB activation. Western blot analysis revealed the presence of inhibitory protein I-kappaBalpha in resting human aortic SMC, which was rapidly phosphorylated and degraded on exposure to interleukin-1beta (IL-1beta) followed by NF-kappaB translocation to the nucleus. IL-1beta had no effect on two measures of intracellular oxidant stress, fluorescence generated by the oxidation of 2',7'-dichlorodihydrofluorescin to dichlorofluorescein (DCF) or changes in intracellular sulfhydryl content. N-acetylcysteine (NAC) a membrane-permeant antioxidant, which augmented intracellular sulfhydryl content and inhibited H(2)O(2)-induced DCF fluorescence, had no effect on cytokine-induced NF-kappaB activation. In contrast to NAC, the metal chelators pyrrolidine dithiocarbamate and diethyldithiocarbamate attenuated IL-1beta-induced NF-kappaB activation but had no effect on intracellular sulfhydryl content. Treatment of the cells with the oxidant H(2)O(2) caused an increase in DCF fluorescence and decreased intracellular sulfhydryl content but had no effect on I-kappaBalpha or NF-kappaB. In conclusion, this study suggests that oxidant stress may not play a major role in cytokine-induced activation of NF-kappaB in human aortic SMC and that oxidants may not be primary activators of NF-kappaB in these cells.
Asunto(s)
Aorta/metabolismo , Interleucina-1/farmacología , Músculo Liso Vascular/metabolismo , FN-kappa B/fisiología , Estrés Oxidativo/fisiología , Antioxidantes/farmacología , Aorta/citología , Aorta/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Membranas Intracelulares/metabolismo , Metales/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oxidantes/farmacología , Compuestos de Sulfhidrilo/metabolismoRESUMEN
We investigated the effects of endothelin-1 (ET-1) on growth of cultured human coronary artery smooth muscle cells (cSMC). ET-1 alone stimulated DNA synthesis in growth-arrested cSMC as measured by [3H]thymidine incorporation, with a maximum 63 +/- 23% increase above control by 10(-7) M (P < 0.05). ET-1 (10(-7) M) also stimulated increases in cyclin D1 protein levels after 24 h, and in absolute cell number after 4 days. Furthermore, ET-1 stimulated protein synthesis (maximum 73 +/- 32% increase in [3H]leucine incorporation by 10(-7) M (P < 0.05)), as well as triggering intracellular calcium transients in human cSMC, as visualised under fura-2 fluorescence microscopy. The selective ET(A) receptor antagonist BQ123 inhibited the increases in DNA synthesis, cell number, protein synthesis and intracellular calcium concentration in response to ET-1, whereas the ET(B) receptor antagonist BQ788 had no such effects. Furthermore, the ET(B) agonist sarafotoxin 6c had no effect on cSMC DNA synthesis. In addition, co-incubation of ET-1 with threshold concentrations of the growth factors, platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), resulted in pronounced synergistic increases in DNA synthesis over that observed with the factors alone. In conclusion, we have shown that ET-1 stimulates proliferation of human cSMC via the ET(A) receptor and is also a co-mitogen with the growth factors tested. These findings indicate a role for ET-1 in the development of coronary intimal hyperplasia in man.
Asunto(s)
Vasos Coronarios/patología , Endotelina-1/farmacología , Sustancias de Crecimiento/farmacología , Mitosis/efectos de los fármacos , Desarrollo de Músculos , Músculo Liso Vascular/crecimiento & desarrollo , Receptores de Endotelina/metabolismo , Becaplermina , Western Blotting , Calcio/metabolismo , Recuento de Células , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , ADN/biosíntesis , ADN/efectos de los fármacos , Antagonistas de los Receptores de Endotelina , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Cardiopatías/metabolismo , Cardiopatías/patología , Humanos , Líquido Intracelular/metabolismo , Microscopía Fluorescente , Mitosis/genética , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Péptidos Cíclicos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Biosíntesis de Proteínas , Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis , Receptor de Endotelina A , Proteínas Recombinantes , TimidinaRESUMEN
1. Interstitial fibroblast proliferation is an elemental feature in the development of cardiac fibrosis. The effects of inhibitors of the intracellular signalling proteins, MEK, a kinase involved in the mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3-K), were tested on growth of cultured human cardiac fibroblasts. 2. Cardiac fibroblasts were isolated from transplant recipient myocardium and made quiescent by serum deprivation for 48 h. Cells were incubated for 24 h with the inhibitors PD 098059 (0.3-30 mumol/L) and LY294002 (1-25 mumol/L) in the presence and absence of platelet-derived growth factor-AB (PDGF-AB, 10 ng/mL). DNA synthesis was measured by [3H]-thymidine incorporation assay (20-24 h). 3. Both compounds markedly inhibited both basal and PDGF-stimulated increases in DNA synthesis in a concentration-dependent manner. Cardiac fibroblast DNA synthesis was reduced to near control levels by PD 098059, while it was inhibited completely by LY294002. 4. These results implicate the importance of MAPK and PI3-K activation in the signal transduction pathways necessary for cardiac fibroblast replication.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Corazón/efectos de los fármacos , Miocardio/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Cromonas/farmacología , Medio de Cultivo Libre de Suero , Inhibidores Enzimáticos/farmacología , Fibroblastos , Fibrosis , Flavonoides/farmacología , Humanos , Morfolinas/farmacología , Miocardio/enzimologíaRESUMEN
We studied the endothelin receptors mediating contraction in the human saphenous vein (SV) and internal thoracic artery (ITA). In the SV, the ET(A)-receptor antagonist BQ123 (1 microM) did not significantly shift the ET-1 concentration-response curve but did cause a parallel shift in the ITA. In the SV, the ET(A)-receptor agonist sarafotoxin 6b (S6b) produced a monophasic concentration-response curve that was antagonised biphasically by BQ123 (0.1-1 microM). In the ITA, S6b was an ineffective agonist with contractions seen only at 3 x 10(-9) M upward. The ET(B)-receptor agonist sarafotoxin 6c (S6c) caused constrictions in only 74% of SV rings and 42% of ITA rings. In the tissues that did respond, S6c caused a monophasic concentration-response curve with a lower maximal response than ET-1. The ET(B) antagonist BQ788 did not antagonise the responses to ET-1 in either the SV or the ITA but did antagonise the responses to S6c in the SV. The results from this study suggest that mainly ET(A) receptors mediate the contractile responses in the human SV and ITA. There is also evidence for an ET(B)-mediated response, although the contractions were much smaller than those elicited by ET-1. We also conclude that the ET(A) receptors mediating responses in these human vessels are atypical because of the different effects of BQ123 on the two vessels.
Asunto(s)
Arterias Mamarias/metabolismo , Receptores de Endotelina/metabolismo , Vena Safena/metabolismo , Adulto , Anciano , Antagonistas de los Receptores de Endotelina , Endotelina-1/farmacología , Humanos , Arterias Mamarias/efectos de los fármacos , Arterias Mamarias/fisiología , Persona de Mediana Edad , Péptidos Cíclicos/farmacología , Receptor de Endotelina B , Vena Safena/efectos de los fármacos , Vena Safena/fisiología , Vasoconstricción/efectos de los fármacosRESUMEN
BACKGROUND: Angiotensin II (Ang II) has been implicated in the development of cardiac fibrosis. The aims of the present study were to examine expression and activity of ACE and of angiotensin receptors in human cardiac fibroblasts cultured from dilated cardiomyopathic and ischemic hearts. The effects of Ang II on fibroblasts were also investigated. METHODS AND RESULTS: Human cardiac fibroblasts were cultured from ventricular and atrial myocardium and characterized immunohistochemically. Expression of ACE and the angiotensin AT1 receptor was demonstrated in cardiac fibroblasts by reverse transcriptase-polymerase chain reaction and radioligand binding. Functional ACE activity, measured by radiolabeled substrate conversion assay, was detected in both ventricular (Vmax. Km-1. mg-1, 0.031+/-0.010; n=13) and atrial (0. 034+/-0.012; n=6) fibroblasts. Fibroblast ACE activity was increased after 48 hours of treatment with basic fibroblast growth factor, dexamethasone, and phorbol ester. Ang II did not affect DNA synthesis but stimulated [3H]proline incorporation in cardiac fibroblasts (20.0+/-4.0% increase above control by 10 micromol/L; P<0.05, n=7), which was abolished by losartan 10 micromol/L but not PD123319 1 micromol/L. Ang II also stimulated a rise in intracellular calcium (basal, 56+/-1 nmol/L; Ang II, 355+/-24 nmol/L) via the AT1 receptor, as shown by complete inhibition with losartan. CONCLUSIONS: We have demonstrated expression and activity of ACE and AT1 receptor in cultured human cardiac fibroblasts. In addition, cardiac fibroblasts respond to Ang II with AT1 receptor-mediated collagen synthesis. The presence of local ACE and AT1 receptors in human fibroblasts suggests their involvement in the development of cardiac fibrosis.
Asunto(s)
Miocardio/metabolismo , Peptidil-Dipeptidasa A/biosíntesis , Receptores de Angiotensina/biosíntesis , Calcio/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Fibrosis , Humanos , Miocardio/patologíaRESUMEN
In recent years, the use of the radial artery as a coronary artery bypass graft has enjoyed a revival. This follows the initial disappointing results with the use of this blood vessel experienced by Carpentier and colleagues in the early 1970s. The improvement in the performance of the radial artery is believed to be caused by improved harvesting techniques and the use of vasodilator drugs. However, compared with the other blood vessels used as bypass grafts, little is known about the vascular biology of this artery. The reactivity of the smooth muscle and protection offered by the vascular endothelium are known to be important factors that may determine the suitability of different arteries and veins to act as bypass conduits. The aim of this review is to examine how the properties of the vessel wall may contribute to the performance of the radial artery when used as a coronary artery bypass graft.