RESUMEN
The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.
Asunto(s)
Bioensayo , Vasos Sanguíneos/efectos de los fármacos , Mediadores de Inflamación/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Citocinas/inmunología , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Humanos , Masculino , Ratones , Contracción Muscular/inmunología , Músculo Liso Vascular/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Péptido Hidrolasas/análisis , Péptido Hidrolasas/inmunología , Ratas , Receptor de Bradiquinina B2/análisis , Receptor de Bradiquinina B2/inmunología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/inmunologíaRESUMEN
An interferometric biosensor comprised of two layers of porous Si, stacked one on top of the other, is described. A fast Fourier transform (FFT) of the reflectivity spectrum reveals three peaks that correspond to the optical thickness of the top layer, the bottom layer, and both layers together. Binding of immunoglobulin G to a protein A capture probe adsorbed to the surface of the top layer induces changes in reflectivity at the top layer/solution interface. The FFT method allows discrimination of target analyte binding from matrix effects due to nonspecific changes in the analyte solution. The sensor response is shown to be insensitive to the addition of 4000-fold excess sucrose or 80-fold excess bovine serum albumin interferents.
Asunto(s)
Técnicas Biosensibles/métodos , Inmunoglobulina G/química , Interferometría/métodos , Dióxido de Silicio/química , Proteína Estafilocócica A/análisis , Animales , Análisis de Fourier , ConejosRESUMEN
Matrix remodeling plays an important role in the physiological and pathological remodeling of blood vessels. We specifically investigated the role of matrix metalloproteinase (MMP)-9, an MMP induced during arterial remodeling, by assessing the effects of genetic MMP-9 deficiency on major parameters of arterial remodeling using the mouse carotid artery flow cessation model. Compared with remodeling of matched wild-type (WT) arteries, MMP-9 deficiency decreased intimal hyperplasia, reduced the late lumen loss, eliminated the correlation between intimal hyperplasia and geometric remodeling, and led to significant accumulation of interstitial collagen. Biochemical analysis of MMP-9 knockout (KO) arterial tissue and isolated smooth muscle cells (SMCs) confirmed the lack of MMP-9 expression or compensation by other gelatinases. To investigate potential mechanisms for the in vivo observations, we analyzed in vitro effects of MMP-9 deficiency on the migration, proliferation, and collagen gel contracting capacity of aortic SMCs isolated from MMP-9 KO and WT mice. Although proliferation was comparable, we found that MMP-9-deficient cells had not only decreased migratory activity, but they also had decreased capacity to contract collagen compared with WT cells. Thus, MMP-9 appears to be involved not only in degradation, but also in reorganization of a collagenous matrix, both facets being essential for the outcome of arterial remodeling. Our results also establish MMP-9 as an attractive therapeutic target for limiting the effects of pathological arterial remodeling in restenosis and atherosclerosis.
Asunto(s)
Arterias Carótidas/enzimología , Movimiento Celular , Marcación de Gen , Metaloproteinasa 9 de la Matriz/deficiencia , Músculo Liso Vascular/enzimología , Animales , Arterias Carótidas/patología , Estenosis Carotídea/enzimología , Estenosis Carotídea/patología , Movimiento Celular/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática/genética , Gelatina/química , Gelatina/metabolismo , Inmunohistoquímica , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Músculo Liso Vascular/patología , Grado de Desobstrucción Vascular/genéticaRESUMEN
BACKGROUND: Recent observations associate plaque instability with expansive arterial remodeling, suggesting a common driving mechanism. METHODS AND RESULTS: To demonstrate that macrophages, a characteristic of vulnerable plaques, also assist in expansive remodeling, we compared carotid artery remodeling due to formation of experimental macrophage-rich and macrophage-poor lesions in the flow cessation model in hypercholesterolemic apolipoprotein E knockout (ApoE KO) and wild type (WT) mice. After ligation, macrophages started to rapidly accumulate in ApoE KO but not in WT carotid artery lesions. Macrophage-rich ApoE KO intimal lesions grew fast, typically occluding within 14 days, despite a tripling of the vessel area. Outward remodeling of macrophage-rich ApoE KO arteries positively correlated with macrophage area (r2=0.600, P<0.001). To investigate potential mechanisms of macrophage-enabled expansive remodeling, we compared levels of matrix metalloproteinases in homogenates of macrophage-rich and macrophage-poor carotid arteries. Gelatinolytic activity of macrophage-rich lesions increased faster and reached maximal levels several fold higher than in the macrophage-poor WT lesions. CONCLUSIONS: Our results suggest that macrophages facilitate expansive arterial remodeling through increased matrix degradation by matrix metalloproteinases. This initially favorable remodeling action may eventually increase the vulnerability of macrophage-rich atherosclerotic plaques.