RESUMEN
Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.
Asunto(s)
Fibrina/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Alantoides/irrigación sanguínea , Alantoides/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Permeabilidad Capilar/efectos de los fármacos , Embrión de Pollo , Corion/irrigación sanguínea , Corion/efectos de los fármacos , Difusión , Implantes de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Endotelio Vascular/citología , Proteínas de la Matriz Extracelular/análisis , Fibrina/administración & dosificación , Geles , Humanos , Ratones , Morfogénesis/efectos de los fármacos , Cadenas Pesadas de Miosina , Miosina Tipo IIB no Muscular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Receptor TIE-2/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/análisis , alfa 2-Antiplasmina/química , alfa 2-Antiplasmina/genéticaRESUMEN
Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.
Asunto(s)
Riñón/irrigación sanguínea , Riñón/embriología , Neovascularización Fisiológica , Animales , Embrión de Pollo , Molde por Corrosión , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Inmunohistoquímica , Riñón/ultraestructura , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/embriología , Glomérulos Renales/ultraestructura , Microcirculación , Microscopía Electrónica , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In contrast to sprouting angiogenesis, which is a well established mode of new blood vessel formation, intussusceptive angiogenesis (IA) is a relatively new concept in vascular biology. It was first discovered in the lung as a means of capillary network growth (intussusceptive microvascular growth). The mechanism consists in the repeated insertion of new slender transcapillary tissue pillars, which subsequently increase in size, thus allowing the capillary network to grow in itself (i.e., by intussusception). It could be shown that IA was present in all organs and species investigated so far, so that it appears to be an ubiquitous phenomenon in vertebrates at least. It was not a surprise therefore to find that IA also played a role in tumour vascularisation. Morphological analysis has yet brought evidence for 6 different modes of pillar formation. They all have in common that, at one time, two endothelial leaflets (e.g. of opposite capillary walls) come into close contact, form new junctional complexes, then thin out to finally give way to the invading interstitial tissue, particularly to fibroblasts, myofibroblasts and pericytes. Once such a transcapillary pillar is formed, it can subsequently grow to the size of a normal intercapillary mesh. The addition of collagen fibrils to the pillar core will stabilize the pillar mechanically. Recent observations allowed to extend the IA concept further: The same structural mechanism of intussusceptive pillar formation was shown to contribute also to the formation of vascular trees (arborisation) and to be involved in vascular remodeling. Although numerous growth factors and receptors have already been suggested as being active in IA, very few hard facts are at present available which would allow to get a comprehensive view of IA regulation.
Asunto(s)
Neovascularización Fisiológica/fisiología , Animales , Capilares/ultraestructura , Embrión de Pollo , Humanos , Pulmón/irrigación sanguínea , Pulmón/crecimiento & desarrollo , Microscopía Electrónica , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Especificidad de Órganos , Ratas , Especificidad de la Especie , Vertebrados/fisiologíaRESUMEN
The metastatic potential of tumors is dependent on the ability of tumor cells to degrade extracellular matrix components by the expression of matrix metalloproteinases (MMPs) and to induce vascularisation of the tumor tissue. Thus, expression of MMPs and the number of blood vessel in tumor tissue may serve as prognostic markers of aggressive and metastasizing tumor growth. We have determined the vascularisation and the expression of MMP-2 by immuno-histochemical staining of 19 benign and 75 malignant breast tissue specimens with CD31- and MMP-2 specific antisera. The degree of vascularisation was expressed by intratumoral microvascular density (IMD), which takes into account all vessels present in a hot spot irrespective of their size. In addition, we have introduced a novel parameter, vascular grading (VG), which describes the percentage of small microvessels of <20 microm in diameter in the total number of blood vessels. IMD tended to indicate an elevated risk for metastasis formation and disease recurrence, while VG did not correlate with metastasis formation. Similarly, MMP-2 expression neither correlated with the clinical outcome of the disease nor with the classical histo-pathological parameters such as stage, grade, lymph node involvement and estrogen receptor status. Tumor cell-specific MMP-2 expression, however, showed a highly significant correlation with VG but not with IMD. These results indicate that MMP-2 expression is rather involved in the formation of small capillaries than in vessel maturation and tumor cell invasion. Thus, MMP-2 expression by tumor cells may serve as indicator of strong angiogenic induction potential of breast tumor cells.
Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Neovascularización Patológica/patología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Biomarcadores de Tumor , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/irrigación sanguínea , Carcinoma Ductal de Mama/enzimología , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/irrigación sanguínea , Carcinoma Lobular/enzimología , Carcinoma Lobular/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Técnicas para Inmunoenzimas , Microcirculación , Invasividad Neoplásica , Estadificación de Neoplasias , Receptores de Estrógenos/metabolismo , Células Tumorales CultivadasRESUMEN
Roughly 90% of the gas-exchange surface is formed by alveolarization of the lungs. To the best of our knowledge, the formation of new alveoli has been followed in rats only by means of morphological description or interpretation of semiquantitative data until now. Therefore, we estimated the number of alveoli in rat lungs between postnatal days 4 and 60 by unambiguously counting the alveolar openings. We observed a bulk formation of new alveoli between days 4 and 21 (17.4 times increase from 0.8 to 14.3 millions) and a second phase of continued alveolarization between days 21 and 60 (1.3 times increase to 19.3 million). The (number weighted) mean volume of the alveoli decreases during the phase of bulk alveolarization from â¼593,000 µm(3) at day 4 to â¼141,000 µm(3) at day 21, but increases again to â¼298,000 µm(3) at day 60. We conclude that the "bulk alveolarization" correlates with the mechanism of classical alveolarization (alveolarization before the microvascular maturation is completed) and that the "continued alveolarization" follows three proposed mechanisms of late alveolarization (alveolarization after microvascular maturation). The biphasic pattern is more evident for the increase in alveolar number than for the formation of new alveolar septa (estimated as the length of the free septal edge). Furthermore, a striking negative correlation between the estimated alveolar size and published data on retention of nanoparticles was detected.
Asunto(s)
Animales Recién Nacidos/crecimiento & desarrollo , Alveolos Pulmonares/crecimiento & desarrollo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The human lung is born with a fraction of the adult complement of alveoli. The postnatal stages of human lung development comprise an alveolar stage, a stage of microvascular maturation, and very likely a stage of late alveolarization. The characteristic structural features of the alveolar stage are well known; they are very alike in human and rat lungs. The bases for alveolar formation are represented by immature inter-airspace walls with two capillary layers with a central sheet of connective tissue. Interalveolar septa are formed by folding up of one of the two capillary layers. In the alveolar stage, alveolar formation occurs rapidly and is typically very conspicuous in both species; it has therefore been termed 'bulk alveolarization'. During and after alveolarization the septa with double capillary networks are restructured to the mature form with a single network. This happens in the stage of microvascular maturation. After these steps the lung proceeds to a phase of growth during which capillary growth by intussusception plays an important role in supporting gas exchange. In view of reports that alveoli are added after the stage of microvascular maturation, the question arises whether the present concept of alveolar formation needs revision. On the basis of morphological and experimental findings we can state that mature lungs contain all the features needed for 'late alveolarization' by the classical septation process. Because of the high plasticity of the lung tissues, late alveolarization or some forms of compensatory alveolar formation may be considered for the human lung.
Asunto(s)
Pulmón/anatomía & histología , Pulmón/crecimiento & desarrollo , Morfogénesis , Alveolos Pulmonares/crecimiento & desarrollo , Animales , Diferenciación Celular , Humanos , Pulmón/irrigación sanguínea , Pulmón/citología , Alveolos Pulmonares/anatomía & histología , Alveolos Pulmonares/irrigación sanguínea , Alveolos Pulmonares/citología , Factores de TiempoRESUMEN
Postnatal glucocorticoid treatment of preterm infants was mimicked by treating newborn rats with dexamethasone (0.1-0.01 microg/g, days 1-4). This regimen has been shown to cause delayed alveolarization. Knowing that microvascular maturation (transformation of double- to single-layered capillary networks in alveolar septa) and septal thinning prevent further alveolarization, we measured septal maturation on electron photomicrographs in treated and control animals. In treated rats and before day 10, we observed a premature nonreversing microvascular maturation and a transient septal thinning, which both appeared focally. In vascular casts of both groups, we observed contacts between the two capillary layers of immature alveolar septa, which were predictive for capillary fusions. Studying serial electron microscopic sections of human lungs, we were able to confirm the postulated fusion process for the first time. We conclude that alveolar microvascular maturation indeed occurs by capillary fusion and that the dexamethasone-induced impairment of alveolarization is associated with focal premature capillary fusion.
Asunto(s)
Animales Recién Nacidos/crecimiento & desarrollo , Dexametasona/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Alveolos Pulmonares/irrigación sanguínea , Alveolos Pulmonares/efectos de los fármacos , Animales , Animales Recién Nacidos/fisiología , Capilares/efectos de los fármacos , Capilares/crecimiento & desarrollo , Capilares/ultraestructura , Femenino , Humanos , Lactante , Masculino , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
Morphometric investigations using a point and intersection counting strategy in the lung often are not able to reveal the full set of morphologic changes. This happens particularly when structural modifications are not expressed in terms of volume density changes and when rough and fine surface density alterations cancel each other at different magnifications. Making use of digital image processing, we present a methodological approach that allows to easily and quickly quantify changes of the geometrical properties of the parenchymal lung structure and reflects closely the visual appreciation of the changes. Randomly sampled digital images from light microscopic sections of lung parenchyma are filtered, binarized, and skeletonized. The lung septa are thus represented as a single-pixel wide line network with nodal points and end points and the corresponding internodal and end segments. By automatically counting the number of points and measuring the lengths of the skeletal segments, the lung architecture can be characterized and very subtle structural changes can be detected. This new methodological approach to lung structure analysis is highly sensitive to morphological changes in the parenchyma: it detected highly significant quantitative alterations in the structure of lungs of rats treated with a glucocorticoid hormone, where the classical morphometry had partly failed.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Pulmón/anatomía & histología , Animales , Artefactos , Dexametasona/farmacología , Glucocorticoides/farmacología , Pulmón/efectos de los fármacos , Masculino , Distribución Aleatoria , RatasRESUMEN
The theory of bifurcating vascular systems predicts vessel diameters that are related to optimality criteria like minimization of pumping energy or of building material. However, mechanisms for producing the postulated optimality have not been described so far, and quantitative data on bifurcation diameters during development are scarce. We used an embryonic vascular bed that rapidly grows and adapts to changing hemodynamic conditions, the chicken chorioallantoic membrane (CAM), and correlated vascular cast and tissue section morphology with in vivo time-lapse video monitoring. The bifurcation exponent delta and associated parameters were quantitatively assessed in arterial and venous microvessels ranging in diameter from 30 to 100 microm. We observed emergence of optimality by means of intussusception, i.e., formation of transvascular tissue pillars. In addition to intussusceptive microvascular growth (IMG = expansion of capillary networks) and intussusceptive arborization (IAR = formation of feeding vessels from capillaries) the observed intussusception at bifurcations represents a third variant of nonsprouting angiogenesis. We call it intussusceptive branching remodeling (IBR). IBR occurred in vessels of considerable diameter by means of two alternative mechanisms: either through pillars arising close to a bifurcation, which increased in girth until they merged with the connective tissue in the bifurcation angle; or through pillars arising at some distance from the bifurcation point, which then expanded by formation of ingrowing tissue folds until they became connected to the tissue of the bifurcation angle. Morphologic evidence suggests that IBR is a wide-spread phenomenon, taking place also in lung, intestinal, kidney, eye, etc., vasculature. Irrespective of the mode followed, IBR led to a branching pattern close to the predicted optimum, delta = 3.0. Significant differences were observed between delta at arterial bifurcations (2.70 to 2.90) and delta at venous bifurcations (2.93 to 3.75). IBR, by means of eccentric pillar formation and fusion, was also involved in vascular pruning. Experimental changes in CAM hemodynamics (by locally increasing blood flow) induced onset of IBR within less than 1 hr. Our study provides morphologic and quantitative evidence that a similar cellular machinery is used for all three variants of vascular intussusception, IMG, IAR, and IBR. It thus provides a mechanism of efficiently generating complex blood transport systems from limited genetic information. Differential quantitative outcome of IBR in arteries and veins, and the experimental induction of IBR strongly suggest that hemodynamic factors can instruct embryonic vascular remodeling toward optimality.
Asunto(s)
Alantoides/irrigación sanguínea , Vasos Sanguíneos/embriología , Corion/irrigación sanguínea , Neovascularización Fisiológica , Alantoides/ultraestructura , Animales , Velocidad del Flujo Sanguíneo , Vasos Sanguíneos/ultraestructura , Embrión de Pollo , Corion/ultraestructura , Molde por Corrosión , Hemodinámica , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/ultraestructura , Modelos Anatómicos , Flujo Sanguíneo RegionalRESUMEN
Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.
Asunto(s)
Envejecimiento/fisiología , Animales Recién Nacidos/anatomía & histología , Animales Recién Nacidos/fisiología , Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Pulmón/fisiología , Animales , División Celular/efectos de los fármacos , Pulmón/citología , Pulmón/crecimiento & desarrollo , Ratas , Ratas Sprague-Dawley , Transglutaminasas/metabolismoRESUMEN
UNLABELLED: Glucocorticoids (GC) are successfully applied in neonatology to improve lung maturation in preterm born babies. Animal studies show that GC can also impair lung development. In this investigation, we used a new approach based on digital image analysis. Microscopic images of lung parenchyma were skeletonised and the geometrical properties of the septal network characterised by analysing the 'skeletal' parameters. Inhibition of the process of alveolarisation after extensive administration of small doses of GC in newborn rats was confirmed by significant changes in the 'skeletal' parameters. The induced structural changes in the lung parenchyma were still present after 60 days in adult rats, clearly indicating a long lasting or even definitive impairment of lung development and maturation caused by GC. CONCLUSION: digital image analysis and skeletonisation proved to be a highly suited approach to assess structural changes in lung parenchyma.
Asunto(s)
Dexametasona/toxicidad , Glucocorticoides/toxicidad , Pulmón/efectos de los fármacos , Factores de Edad , Animales , Animales Recién Nacidos , Procesamiento de Imagen Asistido por Computador , Pulmón/crecimiento & desarrollo , Pulmón/patología , Masculino , Microscopía por Video/métodos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/patología , Ratas , Factores de TiempoRESUMEN
During most instances of angiogenesis, not only are the capillaries or terminal vessels generated and modified, but the supplying vascular system is subjected to remodeling as well. Intussusception, i.e., transluminal pillar formation, is one essential mechanism for growth, arborization, bifurcation remodeling, and pruning. Complex and efficient vascular beds can thus be generated by local interactions between vascular cells and hemodynamic conditions.
Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Neovascularización Fisiológica/fisiología , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Capilares/crecimiento & desarrollo , Capilares/fisiología , Hemodinámica/fisiología , Humanos , Modelos BiológicosRESUMEN
Intussusception (growth within itself) is an alternative to the sprouting mode of angiogenesis. The protrusion of opposing microvascular walls into the capillary lumen creates a contact zone between endothelial cells. The endothelial bilayer is perforated, intercellular contacts are reorganized, and a transluminal pillar with an interstitial core is formed, which is soon invaded by myofibroblasts and pericytes leading to its rapid enlargement by the deposition of collagen fibrils. Intussusception has been implicated in three processes of vascular growth and remodeling. (1) Intussusceptive microvascular growth permits rapid expansion of the capillary plexus, furnishing a large endothelial surface for metabolic exchange. (2) Intussusceptive arborization causes changes in the size, position, and form of preferentially perfused capillary segments, creating a hierarchical tree. (3) Intussusceptive branching remodeling (IBR) leads to modification of the branching geometry of supplying vessels, optimizing pre- and postcapillary flow properties. IBR can also lead to the removal of branches by pruning in response to changes in metabolic needs. None of the three modes requires the immediate proliferation of endothelial cells but rather the rearrangement and plastic remodeling of existing ones. Intussusception appears to be triggered immediately after the formation of the primitive capillary plexus by vasculogenesis or sprouting. The advantage of this mechanism of growth over sprouting is that blood vessels are generated more rapidly in an energetically and metabolically more economic manner, as extensive cell proliferation, basement membrane degradation, and invasion of the surrounding tissue are not required; the capillaries thereby formed are less leaky. This process occurs without disrupting organ function. Improvements in our understanding of the process should enable the development of novel pro- and anti-angiogenic therapeutic treatments.
Asunto(s)
Vasos Sanguíneos/embriología , Capilares/embriología , Neovascularización Fisiológica , Alantoides/irrigación sanguínea , Animales , Vasos Sanguíneos/ultraestructura , Capilares/ultraestructura , Embrión de Pollo , Corion/irrigación sanguínea , Corion/embriología , Corion/ultraestructura , Molde por Corrosión , HumanosRESUMEN
This review shall familiarize the reader with the various aspects of intussusceptive angiogenesis (IA). The basic event in IA is the formation of transvascular tissue pillars. Depending on location, timing, and frequency of pillar emergence, the IA process has different outcomes. In capillaries, a primary IA function is to expand the capillary bed in size and complexity (intussusceptive microvascular growth). It represents an alternative to capillary sprouting. Highly ordered pillar formation in a developing capillary network leads to the formation of vascular trees (intussusceptive arborization). In small arteries and veins, pillar formation at the vessels' branching angles leads either to remodeling of the branching geometry or even to vascular pruning (intussusceptive branching remodeling). It appears essential that future angiogenic research considers always both phenomena, sprouting and intussusception. Vascularization of tissues, organs, and tumors rely heavily on both mechanisms; neglecting one or the other would obscure our understanding of the angiogenesis process.
Asunto(s)
Vasos Sanguíneos/embriología , Capilares/embriología , Neovascularización Fisiológica , Animales , Vasos Sanguíneos/ultraestructura , Capilares/ultraestructura , Embrión de Pollo , Corion/irrigación sanguínea , Corion/ultraestructura , Molde por Corrosión , Humanos , Microcirculación , Microscopía Electrónica de Rastreo , Modelos BiológicosRESUMEN
While glucocorticoid (GC) administration appears to be beneficial during the acute phase of treatment of neonates at risk of developing chronic lung disease, it is still not clear whether steroid application has an adverse long-term effect on the lung maturation. Thus, the goal of the present work was to analyze GC effects on the pulmonary structure in a rat model where dosage and timing of drug administration were adapted to the therapeutic situation in human neonatology. The animals received daily a maximum of 0.1 mg dexamethasone phosphate per kilogram body weight during the first 4 postnatal days. Investigations were performed at the light microscopic level by means of a digital image analysis system. While there were no differences in the lung architecture between experimental animals and controls on day 4, the earliest time point of observation, we found a widening of airspaces with a concomitant decrease in the alveolar surface area density, representing a loss of parenchymal complexity, on days 10 and 21 in treated rats. On days 36 and 60, however, no alterations in the pulmonary parenchyma could be detected in experimental animals. We conclude from these findings that the GC-induced initial inhibition of development (days 10 and 21) was completely reversed, so that a normal parenchymal architecture and also a normal alveolar surface area density were found in adult rats (days 36 and 60). From the results obtained using the regimen of GC administration described, mimicking more closely the steroid treatment in human neonatology, we conclude that the observed short-term adverse effects on lung development can be fully compensated until adult age.
Asunto(s)
Animales Recién Nacidos/fisiología , Dexametasona/administración & dosificación , Dexametasona/efectos adversos , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Pulmón/patología , Adaptación Fisiológica , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Procesamiento de Imagen Asistido por Computador , Pulmón/efectos de los fármacos , Masculino , RatasRESUMEN
It has been shown that glucocorticoids accelerate lung development by limiting alveolar formation resulting from a premature maturation of the alveolar septa. Based on these data, the aim of the present work was to analyze the influence of dexamethasone on cell cycle control mechanisms during postnatal lung development. Cell proliferation is regulated by a network of signaling pathways that converge to the key regulator of cell cycle machinery: the cyclin-dependent kinase (CDK) system. The activity of the various cyclin/CDK complexes can be modulated by the levels of the cyclins and their CDKs, and by expression of specific CDK inhibitors (CKIs). In the present study, newborn rats were given a 4-d treatment with dexamethasone (0.1-0.01 microg/g body weight dexamethasone sodium phosphate daily on d 1-4), or saline. Morphologically, the treatment caused a significant thinning of the septa and an acceleration of lung maturation on d 4. Study of cyclin/CDK system at d 1-36 documented a transient down-regulation of cyclin/CDK complex activities at d 4 in the dexamethasone-treated animals. Analysis of the mechanisms involved suggested a role for the CKIs p21CIP1 and p27KIP1. Indeed, we observed an increase in p21CIP1 and p27KIP1 protein levels on d 4 in the dexamethasone-treated animals. By contrast, no variations in either cyclin and CDK expression, or cyclin/CDK complex formation could be documented. We conclude that glucocorticoids may accelerate lung maturation by influencing cell cycle control mechanisms, mainly through impairment of G1 cyclin/CDK complex activation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/crecimiento & desarrollo , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Recién Nacidos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Femenino , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Ratas , Ratas Endogámicas , Extractos de Tejidos/química , Extractos de Tejidos/metabolismoRESUMEN
Glucocorticoids are often applied in neonatology and perinatology to fight the problems of respiratory distress and chronic lung disease. There are, however, many controversies regarding the adverse side effects and long-term clinical benefits of this therapeutic approach. In rats, glucocorticoids are known to seriously impair the formation of alveoli when applied during the first two postnatal weeks even at very low dosage. The current study investigates short-term and long-term glucocorticoid effects on the rat lung by means of morphologic and morphometric observations at light and electron microscopic levels. Application of a high-dosage protocol for only few days resulted in a marked acceleration of lung development with a precocious microvascular maturation resulting in single capillary network septa in the first 4 postnatal days. By postnatal d 10, the lung morphologic phenotype showed a step back in the maturational state, with an increased number of septa with double capillary layer, followed by an exceptional second round of the alveolarization process. As a result of this process, there was an almost complete recovery in the parenchymal lung structure by postnatal d 36, and by d 60, there were virtually no qualitative or quantitative differences between experimental and control rats. These findings indicate that both dosage and duration of glucocorticoid therapy in the early postnatal period are very critical with respect to lung development and maturation and that a careful therapeutic strategy can minimize late sequelae of treatment.
Asunto(s)
Animales Recién Nacidos , Glucocorticoides/farmacología , Pulmón/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Femenino , Pulmón/crecimiento & desarrollo , Pulmón/ultraestructura , Microscopía Electrónica , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To evaluate the effects of a 60% vitamin A deficiency (VAD) on the two postnatal stages of lung development: alveolarization and microvascular maturation. Lungs from deficient rats were compared to age-matched controls. STUDY DESIGN: Starting at 3 weeks before mating, female rats were maintained under a diet lacking vitamin A. Due to the slow depletion of the vitamin A liver stores the pregnant rats carried to term and delivered pups under mild VAD conditions. Mothers and offspring were then kept under the same diet what resulted in a mean reduction of vitamin A plasma concentration of about 60% vs. controls during the whole experimental period. Pups were sacrificed on days 4, 10 and 21 and their lungs fixed and analyzed by means of a combined morphologic and morphometric investigation at light and electron microscopic levels. RESULTS: During the whole experiment, body weights of VAD animals were lower than controls with a significant decrease on day 10. On days 4, 10 and 21 the pulmonary structure was in a comparable gross morphologic state in both groups. Despite this morphologic normality, quantitative alterations in some functional parameters could be detected. On day 4, lung volume and the volume and surface area of air spaces were decreased, while the arithmetic mean barrier thickness and type 2 pneumocyte volume were increased in the VAD group. On day 21, some changes were again manifest mainly consisting in an augmentation of the vascularization and a decrease in interstitial volume in deficient animals. CONCLUSIONS: Mild VAD causes no gross disturbances in the postnatal phases of lung development in rats. However, a body weight-related transient retardation of lung maturation was detectable in the first postnatal week. At 3 weeks, the VAD lungs showed a more mature vascular system substantiated by an increase in volume of both capillary volume and the large non-parenchymal vessels. In view of these quantitative alterations, we suspect that mild VAD deregulates the normal phases of body and lung growth, but does not induce serious functional impairments.