RESUMEN
The role of endothelin in oxygen-induced contraction remains controversial. The present study was designed to investigate the role of endothelin in O2-induced contraction in the isolated ductal preparation of rabbit and rat, using the endothelin receptor antagonists, bosentan (antagonist for both ET-A and ET-B receptors) and BQ 485 (an ET-A selective antagonist). The ductus was isolated from fetal rabbits at 30 days of gestation (term 31 days) and fetal rats at 21 days of gestation (term 22 days). In the rabbit ductus with intact endothelium, 13% of the O2-induced contraction was inhibited by bosentan and 14% by BQ 485. In the rabbit ductus without endothelium, bosentan did not cause significant inhibition of the oxygen-induced contraction. In the rat ductus with intact endothelium, about 44% of the O2-induced contraction was inhibited by bosentan. In the rat ductus without endothelium, O2-induced contraction was 20% less than that in the ductus with intact endothelium. In the rat ductus without endothelium, bosentan further decreased the oxygen-induced contraction by about 24%. These data suggest that (1) endothelin plays a significant role in O2-induced contraction in the isolated ductus arteriosus, (2) there is a species difference in the degree of contribution of endothelin to the O2-induced ductal contraction, and (3) there is a species difference in the degree of contribution of the endothelium and vascular smooth muscle cells to the release of endothelin from the ductus arteriosus.
Asunto(s)
Conducto Arterial/efectos de los fármacos , Endotelinas/fisiología , Oxígeno/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Antihipertensivos/farmacología , Azepinas/farmacología , Bosentán , Conducto Arterial/química , Conducto Arterial/fisiología , Antagonistas de los Receptores de la Endotelina A , Antagonistas de los Receptores de la Endotelina B , Endotelinas/análisis , Endotelio/química , Endotelio/efectos de los fármacos , Endotelio/fisiología , Femenino , Inmunohistoquímica , Técnicas In Vitro , Masculino , Oligopéptidos/farmacología , Conejos , Ratas , Receptor de Endotelina A/fisiología , Receptor de Endotelina B/fisiología , Sulfonamidas/farmacología , Vasoconstricción/fisiologíaRESUMEN
We compared the total density and the relative expression of EP receptor (EP) subtypes in ductus arteriosus (DA) of the newborn with that of the fetal piglet. Saturation binding experiments showed 3-fold less PGE2 receptors in the newborn than in the fetus because of loss of EP3 and EP4 receptors thus explaining, at least partly, the reduced responsiveness to PGE2 of the newborn DA. Displacement experiments showed that the relative proportions of EP2, EP3, and EP4 were similar in the fetal DA but only EP2 was detected in the DA of the newborn pig. Hence, PGE2 effects in the newborn DA seem to be exclusively mediated by EP2 receptors both in vitro and in vivo. These findings may help to propose more specific therapies for regulation of DA's tone in certain newborns for whom conventional therapy is contraindicated.
Asunto(s)
Animales Recién Nacidos/metabolismo , Conducto Arterial/química , Conducto Arterial/fisiología , Feto/metabolismo , Receptores de Prostaglandina E/fisiología , Animales , AMP Cíclico/biosíntesis , Dinoprostona/metabolismo , Dinoprostona/farmacología , Conducto Arterial/efectos de los fármacos , Receptores de Prostaglandina E/análisis , Receptores de Prostaglandina E/efectos de los fármacos , Porcinos , TritioRESUMEN
Apoptosis regulates the remodeling of tissue during embryonic development by eliminating unwanted cells and structures. The present study investigated smooth muscle cell (SMC) proliferation and apoptosis in the neonatal ductus arteriosus (DA) during closure. In the DA of 39 swine neonates and 5 autopsy human neonates, apoptosis was detected using in situ end-labeling and electron microscopy, and proliferation was evaluated using proliferating cell nuclear antigen. In swine, apoptosis of SMC was first observed at 24h after birth. After 48h, both apoptosis and proliferation quickly increased and became most prominent at 3 days, mainly in the intima and inner media. From 5 days, both apoptosis and proliferation quickly disappeared, and were present to a minor extent at the 2 weeks after birth. During these processes, there was no sign of inflammation or necrosis. In humans, apoptosis was found in tissue specimens obtained from 2 term neonates who died at 1 and 5 days after birth. These findings suggest that SMC contribute to the functional closure of the DA by active constriction, and soon after, they switch to proliferation and apoptosis, which may contribute to the anatomical closure of the DA.
Asunto(s)
Apoptosis/fisiología , Conducto Arterial/citología , Animales , Animales Recién Nacidos , División Celular , Conducto Arterial/química , Conducto Arterial/fisiología , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Recién Nacido , Microscopía Electrónica , Músculo Liso Vascular/citología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Porcinos , Factores de TiempoRESUMEN
To verify that apoptosis is one of the possible mechanisms of neonatal vascular remodeling during the transition from fetal to neonatal circulation, we assayed for apoptosis and evaluated the expression of apoptosis-regulatory proteins in umbilical vessel versus ascending aorta, ductus arteriosus (DA) versus adjacent pulmonary artery and aorta, or aorta versus its branching arteries. Twenty-two umbilical cords (UCs), 6 DAs with adjacent aortas and pulmonary arteries, and 4 aortic arches with their branching great arteries were obtained from neonates. Smooth muscle cell (SMC) apoptosis in umbilical vessels was identified in all UCs. The expressions of Bax and Bcl-X were stronger in umbilical artery than in the neonatal aorta, but Bcl-2 was weak in both arteries in immunohistochemistry. In the immunoblot analysis of UCs, the expression of the proapoptotic short isoform of Bcl-X was stronger than in other tissue, and caspase-3 was selectively activated, whereas it was not in the other components of the cardiovascular system. In contrast, the expression patterns of the FasAg and Fas ligand were similar in umbilical artery and aorta. Regulation of Bcl-2 family proteins was also observed in other vascular sites at which SMCs undergo apoptosis on hemodynamic changes during birth, such as the DA and the branching points of the great arteries from the aortic arch. Apoptosis is involved in the regression of human umbilical vessels and the DA and in the remodeling of the branching great arteries during the neonatal period, when Bcl-2 family proteins are likely to play a key role.
Asunto(s)
Apoptosis , Vasos Sanguíneos/citología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas/análisis , Aorta/química , Aorta/citología , Vasos Sanguíneos/química , Caspasa 3 , Caspasas/metabolismo , Fragmentación del ADN , Conducto Arterial/química , Conducto Arterial/citología , Activación Enzimática , Humanos , Etiquetado Corte-Fin in Situ , Recién Nacido , Músculo Liso Vascular/anatomía & histología , Músculo Liso Vascular/citología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Arteria Pulmonar/química , Arteria Pulmonar/citología , Cordón Umbilical/química , Cordón Umbilical/citología , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Our studies and those of others have shown that changes in the extracellular matrix have profound effects on vascular remodeling. In the ductus, increased production of endothelial hyaluronan and smooth muscle cell chondroitin sulfate and fibronectin and impaired elastin fiber assembly are features critical to smooth muscle cell migration into the subendothelium and intimal cushion formation. There is a developmentally orchestrated process that involves post-transcriptional mechanisms of gene regulation. Closure of the ductus arteriosus is associated with further changes in matrix expression and programmed cell death. The changes in the extracellular matrix that induce neointimal formation are also observed in pathological conditions in pulmonary and coronary arteries. Plasticity of the pulmonary circulation in the perinatal period also involves matrix regulation, and processes that prevent the normal decrease in pulmonary vascular resistance will result in impaired matrix regulation, and in the development of structural changes in the pulmonary arteries, including abnormal smooth muscle cell differentiation, hypertrophy and proliferation, which sustain the elevation in pulmonary artery pressure.
Asunto(s)
Conducto Arterial/fisiología , Matriz Extracelular/fisiología , Circulación Pulmonar , Animales , Conducto Arterial/química , Conducto Arterial/embriología , Conducto Arterial/ultraestructura , Endotelio Vascular/química , Endotelio Vascular/fisiología , Endotelio Vascular/ultraestructura , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/fisiología , Humanos , Recién Nacido , Arteria Pulmonar/fisiologíaRESUMEN
BACKGROUND: The closure of the ductus arteriosus (DA) is one of the most striking cardiovascular events that occur at birth. It has been attributed to oxygenation and intrinsic prostaglandins. However, selective constriction of DA suggests the presence of highly specialized contractile mechanisms in DA. We previously reported that smooth muscle myosin heavy chain isoforms, SM1 and SM2, are molecular markers for smooth muscle differentiation because of their unique expression pattern during vascular development. SM1 and SM2 are generated from a single gene through developmentally regulated alternative RNA splicing; SM1 is expressed in almost all stages of differentiation of the vascular smooth muscles, but SM2 is found only after birth. METHODS AND RESULTS: Immunohistochemistry was performed to study the expression of the different types of myosin heavy chain isoforms in DA of fetal and neonatal rabbits. Electron microscopic examinations were also carried out to demonstrate ultrastructural characteristics of ductus muscles. We found that SM2 is expressed before birth in the medial layer of DA, indicating advanced differentiation of smooth muscle cells in DA. The exact location of immunoreactivity for SM2 was in the smooth muscle cell of the medial layer of DA. Immunoreactivity for SM1, however, was not different for DA and adjacent great arteries. Transmission electron microscopy demonstrated greater amounts of myofilaments in medial smooth muscles of DA than those of aorta. CONCLUSIONS: These results indicate that smooth muscles in DA are more differentiated than those in other arteries, which may be one of the cellular mechanisms responsible for the unique closure of DA at birth.
Asunto(s)
Conducto Arterial/citología , Músculo Liso Vascular/citología , Miosinas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Animales , Aorta/citología , Diferenciación Celular/fisiología , Conducto Arterial/química , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Músculo Liso Vascular/química , Arteria Pulmonar/citología , Conejos , Arterias Umbilicales/citologíaRESUMEN
BACKGROUND: The ductus arteriosus (DA) is a fetal vessel in which the elastic laminae fail to assemble normally in late gestation. This feature is associated with the development of intimal cushions, structures that partially occlude the DA lumen and assure that the vessel will close completely when it constricts postnatally. EXPERIMENTAL DESIGN: We studied the fetal lamb DA at two different gestational time-points, 100 days before, and 138 days coincident with intimal cushion formation (term = 145 days) to establish the ultrastructural basis for the 'disassembly' of elastic laminae apparent on light microscopy and to determine further whether the mechanism was due to increased elastolytic activity, decreased synthesis of tropoelastin, or impaired insolubilization of tropoelastin. RESULTS: Morphometric ultrastructural analyses of tissue from the 138-day gestation fetal lambs revealed that the volume density of elastin in the DA vessel wall was only 40% of that in the aorta (Ao) and 50% of that in the pulmonary artery (PA). Moreover, only 16% of the elastin present contributed to the formation of laminae when compared to 80% in the Ao and 50% in the PA. Despite the morphologic appearance of 'fragmented' elastin, there was no evidence of increased elastolytic activity in the DA at either gestational time-point as judged by solubilization of a [3H] elastin substrate. The reduced elastin apparent was morphologically accompanied by an increase in soluble (tropo) elastin in DA compared with Ao and PA, as measured by enzyme linked immunosorbent assay, in tissue from both 100- and 138-day gestation lambs. Lack of differences in tropoelastin mRNA levels when comparing the 3 vessels suggested that the enzyme linked immunosorbent assay measurements reflected increased DA tropoelastin accumulation owing to lack of insolubilization rather than an increase in synthesis. Reduced insolubilization of newly synthesized elastin was evident in the DA compared with the Ao at 100 days gestation and in the DA compared with both Ao and PA at 138 days gestation in association with reduced desmosine levels. CONCLUSIONS: The mechanism of the decrease in tropoelastin insolubilization was unrelated to lysyl oxidase activity in the tissue and represents a unique developmental program.
Asunto(s)
Conducto Arterial/embriología , Elastina/metabolismo , Ovinos/embriología , Animales , Aorta/química , Aorta/embriología , Aorta/metabolismo , Membrana Basal/química , Membrana Basal/embriología , Membrana Basal/metabolismo , Conducto Arterial/química , Conducto Arterial/metabolismo , Elastina/análisis , Elastina/genética , Ensayo de Inmunoadsorción Enzimática , Feto/metabolismo , Microscopía Inmunoelectrónica , Elastasa Pancreática/metabolismo , Elastasa Pancreática/fisiología , Proteína-Lisina 6-Oxidasa/fisiología , Arteria Pulmonar/química , Arteria Pulmonar/embriología , Arteria Pulmonar/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Tropoelastina/genética , Tropoelastina/metabolismoRESUMEN
Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in N-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a three-dimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.
Asunto(s)
Sulfatos de Condroitina/farmacología , Elastina/metabolismo , Músculo Liso Vascular/citología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Aorta/química , Adhesión Celular , Movimiento Celular , Células Cultivadas , Sulfatos de Condroitina/análisis , Conducto Arterial/química , Fibronectinas/biosíntesis , Heparitina Sulfato/farmacología , Datos de Secuencia Molecular , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Receptores de Superficie Celular/análisis , OvinosRESUMEN
In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.
Asunto(s)
Sulfatos de Condroitina/farmacología , Conducto Arterial/química , Elastina/análisis , Músculo Liso Vascular/química , Receptores de Superficie Celular/análisis , Animales , Aorta/química , Células Cultivadas , Femenino , Embarazo , OvinosRESUMEN
Changes in distribution of elastin and elastin receptor were studied during maturation of the ductus arteriosus. In this vessel intimal thickening is a normal process. It starts with separation of endothelial cells from the internal elastic lamina by the formation of a wide subendothelial region, followed by an increase in number of radially orientated cells in the inner media and subendothelial region. For the first time the nature of these originally inner media cells could be definitely determined as being derived from smooth muscle cells. Areas rich in these modified smooth muscle cells contained less elastin as compared with regions rich in circularly orientated smooth muscle cells. The internal elastic lamina had disappeared underneath intimal cushions of the canine ductus arteriosus, whereas in the fetal human ductus arteriosus, splitting of the internal elastic lamina was observed. Elastin was not present underneath separated endothelial cells, which suggests that these cells do not synthesize elastin, at least not after separation. Both smooth muscle cells and endothelial cells demonstrated the presence of the elastin receptor. Changes in its distribution were not observed. The temporal and spatial association between the altering distribution of elastin and the absence of normal cushion formation in the persistent ductus arteriosus suggests a role of the elastin receptor in the process of intimal thickening.