Differentiation of vascular endothelium in the chick chorioallantois: a structural and autoradiographic study

The maturation of vascular endothelial cells in the chick chorioallantoic membrane, from 8 to 18 days after fertilization, was investigated by light and electron microscopy. Light microscopic autoradiography following administration of tritiated thymidine was used to determine the thymidine labeling index of the endothelial cell population at various stages of development. Results indicate that, prior to day 11 of incubation, endothelial cells have the morphological characteristics of immature and relatively undifferentiated cells. During this time they exhibit a high labeling index of approximately 23%. At 11 days, the labeling index decreases to 2.8%, and subsequently the cells begin to acquire structural characteristics of mature differentiated endothelium. The pattern of endothelial cell labeling suggests that, during the period of high endothelial cell mitosis, the capillary network of the growing chorioallantoic membrane is expanding by an overall proliferation of endothelial cells in existing capillaries, rather than by formation of new capillary sprouts. The immaturity of endothelial cells in the young chorioallantois, or conversely their high rate of cell division, may influence the ability of this membrane to support grafted tissue prior to day 11.     

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.     

Morphological Ground of Formation of the Brain Choroid Plexus Blood-Cerebrospinal Fluid Barrier in Human Prenatal Ontogenesis

Using light and electron microscopy and immunocytochemistry methods, structural organization of the formed blood-cerebrospinal fluid barrier (BCSFB) of the human brain choroid plexus in embryos of 6–9 weeks of development was studied. The main structures peculiar to the mature BCSFB have been established to appear with formation of the choroid plexus at the end of the 2nd month of the human intrauterine development. Fenestrae in the choroid plexus capillary endothelium are revealed since the 9th week of prenatal development. Characteristic of the human embryonic BCSFB are a poor development of the plexus capillary basal membrane, scanty pericytes, a high activity of interstitial macrophages, which suggests the barrier immaturity. A significant amount of cytoplasmic glycogen inclusions revealed in plexus epitheliocytes seems to be due to peculiar trophic requirements of developing brain cells under conditions of an insufficient development of the local blood supply.     

The development of components of the blood-brain barrier in the neocortex of the white rat

In formation and differentiation of the hematoencephalic barrier elements in white non-inbred rats of 14 age groups (55 animals) at the ultrastructural level certain regular phases of the process have been revealed. For example, morphological maturation of endotheliocytes and pericytes occurs on the 7th day of the postnatal life. Maximal decrease in permeability of the hematoencephalic barrier takes place on the 10th day, as a consequence of liquidation of the pericapillary space between the basal membrane and the glial tunic of the capillaries. Morphofunctional maturation of the hematoencephalic barrier (the 21st day) terminates in differentiation of the pericapillary astrocytic limbs, that surround up to 85% of the capillary perimeter.     

长时间低氧对大鼠颈动脉体培养细胞的细胞内pH和膜电位的影响

本文的目的是研究长时间低氧对离体培养的大鼠颈动脉体球细胞（ｇｌｏｍｕｓｃｅｌｌ）的影响。对实验组Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ（ＳＤ）大鼠,首先将其置于模拟５０００ｍ高度低氧环境的低压舱中饲养７—１０ｄ,然后麻醉动物,取出颈动脉体,将其分离成单个细胞和细胞群体（ｃｌｕｓｔｅｒｓ）。这些细胞在低氧条件（１１％Ｏ２,５％ＣＯ２,８４％Ｎ２）下培养２—３ｄ。取自正常ＳＤ大鼠的颈动脉体细胞被分为两组,分别将其培养在常氧（２１％Ｏ２,５％ＣＯ２,７４％Ｎ２）或低氧环境中。球细胞的细胞内ｐＨ（ｐＨｉ）和膜电位（ＭＰ）分别用Ｈ＋选择性微电极和常规微电极同时测量。结果表明：长时间低氧降低球细胞的ｐＨｉ,增加ＭＰ,其变化程度远远大于急性低氧的影响,而且当将细胞置于常氧中测量时其值不恢复。     

Effects of microbeam radiation therapy on normal and tumoral blood vessels

Microbeam radiation therapy (MRT) is a new form of preclinical radiotherapy using quasi-parallel arrays of synchrotron X-ray microbeams. While the deposition of several hundred Grays in the microbeam paths, the normal brain tissues presents a high tolerance which is accompanied by the permanence of apparently normal vessels. Conversely, the efficiency of MRT on tumor growth control is thought to be related to a preferential damaging of tumor blood vessels.The high resistance of the healthy vascular network was demonstrated in different animal models by in vivo biphoton microscopy, magnetic resonance imaging, and histological studies. While a transient increase in permeability was shown, the structure of the vessels remained intact. The use of a chick chorioallantoic membrane at different stages of development showed that the damages induced by microbeams depend on vessel maturation. In vivo and ultrastructural observations showed negligible effects of microbeams on the mature vasculature at late stages of development; nevertheless a complete destruction of the immature capillary plexus was found in the microbeam paths. The use of MRT in rodent models revealed a preferential effect on tumor vessels. Although no major modification was observed in the vasculature of normal brain tissue, tumors showed a denudation of capillaries accompanied by transient increased permeability followed by reduced tumor perfusion and finally, a decrease in number of tumor vessels. Thus, MRT is a very promising treatment strategy with pronounced tumor control effects most likely based on the anti-vascular effects of MRT.     

Prenatal oxidative stress: I. Malondialdehyde in hypoxic and hyperoxic chick embryos

Evidence suggests a positive correlation between metabolic rate (VO2), or ambient oxygen (O2) tension, and the rate of formation of free radicals from O2. We have previously demonstrated that the rates of growth, VO2, protein and DNA accumulation, and the activity of cytochrome oxidase (a key mitochondrial respiratory enzyme), are increased significantly by exposing the chick embryo to 72 h of hyperoxia (60% O2) late in incubation. To test the hypothesis that the chick embryo responds to a prenatal alteration in O2 availability in such a way as to protect its tissues from oxidative damage, we have used the thiobarbituric acid assay to estimate lipid peroxidation (a major form of free radical damage) in selected organs from chick embryos exposed to altered O2 availability. We found significantly higher concentrations of malondialdehyde (MDA, a secondary product of lipid peroxidation) in liver than in chorioallantoic membrane, brain, or heart. However, embryos exposed to brief (72 h) hypoxia (15% O2) or hyperoxia (60% O2) late in incubation, or 48 h of such exposure followed by 24 h of incubation in pure O2, exhibited no significant difference in MDA levels compared to normoxic (21% O2) controls in any of the tissues examined. We conclude that the increase in aerobic metabolism induced in the chick embryo by 3 days of hyperoxia is not accompanied by an increase in lipid peroxidation. We postulate that the chick embryo adapts to hyperoxia in such a way as to escape additional free radical damage, perhaps by increasing the capacity of its antioxidant defenses to compensate for a potential increase in the rate of free radical generation.     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

Complete development of Isopora suis of swine in chicken embryos

Development of the swine coccidium , Isospora suis, in embryonated chicken eggs is described. The allantoic cavities of eight-to-ten-day-old white Leghorn embryos were inoculated with either 100,000 or 200,000 sporozoites. Developmental stages morphologically similar to those found in the intestines of piglets were present in the endodermal layer of the chorioallantoic membrane (CAM), beginning three days post inoculation (PI). No stages were found in the mesodermal or ectodermal layers of the CAM and none were observed in heart, lung, liver, or spleen. Type I meronts and merozoites were found on days 3 through 10 PI. Type II meronts and merozoites were found days 4 through 10 PI. Mature microgamonts , macrogamonts, and oocysts were found on days 7 through 10 PI. Oocysts appeared to be retained in the endodermal cells and in ovo sporulation did not occur. Attempts to sporulate CAM-derived oocysts were not successful. Isospora suis was not pathogenic for embryos under the conditions of this study. This study represents the first fully documented report of complete development of a mammalian coccidium in chicken embryos.     

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.     

Increase in pulmonary capillary permeability in dogs exposed to 100% O2

Changes in pulmonary capillary filtration induced by hyperoxia were investigated in 15 dogs. After 12 h of normobaric hyperoxic exposure, animals were anesthetized and artificially ventilated with 100% O2. A pulmonary lymphatic vessel was cannulated, and lymph flow and protein content were measured together with pulmonary and systemic hemodynamics. An increase in pulmonary capillary filtration was found when compared with reference data (normoxic dogs in similar conditions) gathered from available literature: lymph flow increased from 21.8 +/- 13.4 to 125.2 +/- 131.6 microliter/min, and the lymph-to-plasma protein concentration ratio increased from 0.67 +/- 0.08 to 0.78 +/- 0.08. To characterize the mechanisms involved, left atrial pressure was increased in two stages (approximately 10 and approximately 25 mmHg). The results clearly indicated an increase in pulmonary capillary permeability as evidenced by a decrease of the minimal estimate of the protein reflection coefficient from 0.62 +/- 0.05 to 0.42 +/- 0.05.     

Developmental Expression of GLUT1 and GLUT3 Glucose Transporters in Rat Brain

Abstract: Two glucose transport proteins, GLUT1 and GLUT3, have been detected in brain. GLUT1 is concentrated in the endothelial cells of the blood-brain barrier and may be present in neurons and glia; GLUT3 is probably the major neuronal glucose transporter. Of the few studies of glucose transport in the immature brain, none has quantified GLUTS. This study used membrane isolation and immunoblotting techniques to examine the developmental expression of GLUT1 and GLUT3 in four forebrain regions, cerebral microvessels, and choroid plexus, from rats 1–30 days postnatally as compared with adults. The GLUT1 level in whole brain samples was low for 14 days, doubled by 21 days, and doubled again to attain adult levels by 30 days; there was no regional variation. The GLUT3 level in these samples was low during the first postnatal week, increased steadily to adult levels by 21–30 days, and demonstrated regional specificity. The concentration of GLUT1 in microvessels increased steadily after the first postnatal week; the GLUT1 level in choroid plexus was high at birth, decreased at 1 week, and then returned to near fetal levels. GLUT3 was not found in microvessels or choroid plexus. This study indicates that both GLUT1 and GLUT3 are developmentally regulated in rat brain: GLUT1 appears to relate to the nutrient supply and overall growth of the brain, whereas GLUT3 more closely relates to functional activity and neuronal maturation.     

Complete Development of Isospora suis of Swine in Chicken Embryos1

Development of the swine coccidium, Isospora suis, in embryonated chicken eggs is described. The allantoic cavities of eight-to-ten-day-old white Leghorn embryos were inoculated with either 100,000 or 200,000 sporozoites. Developmental stages morphologically similar to those found in the intestines of piglets were present in the endodermal layer of the chorioallantoic membrane (CAM), beginning three days post inoculation (PI). No stages were found in the mesodermal or ectodermal layers of the CAM and none were observed in heart, lung, liver, or spleen. Type I meronts and merozoites were found on days 3 through 10 PI. Type II meronts and merozoites were found days 4 through 10 PI. Mature microgamonts, macrogamonts, and oocysts were found on days 7 through 10 PI. Oocysts appeared to be retained in the endodermal cells and in ovo sporulation did not occur. Attempts to sporulate CAM-derived oocysts were not successful. Isospora suis was not pathogenic for embryos under the conditions of this study. This study represents the first fully documented report of complete development of a mammalian coccidium in chicken embryos.     

Influence of maternal diabetes on basement membranes, type 2 cells, and capillaries in the developing rat lung

To determine the effect of maternal diabetes on rat lung development, we studied the ultrastructure of the alveolar wall from the ninteenth day of gestation (term = 22 days) through the eighth postnatal day in fetal and neonatal rats of mothers with streptozotocin-induced diabetes. In normal fetal lung development, epithelial basement membranes develop large discontinuities beneath type 2 cells, through which cytoplasmic foot processes extend into the interstitium. Maternal diabetes delays the appearances of these epithelial basement membrane discontinuities and reduces the number of type 2 cell processes that penetrate it. These alterations in epithelial basement membrane are reversed after birth. There is no ultrastructural evidence of a delay in type 2 cell maturation as assessed by lamellar body volume density morphometry. Endothelial basement membranes, which are not present around the growing pulmonary capillary bed in the pseudoglandular lung, are seen late in normal gestation, primarily around capillaries forming the mature air-blood barrier. This development of endothelial basement membrane may be delayed in the fetuses of diabetic mothers and reflects a significant delay in the expansion of the pulmonary capillary network in these animals as assessed by morphometric volume density measurements. This effect on capillary growth is not reversed in the newborn animals through 8 days after birth. The summation of these effects indicates a generalized slowing of fetal lung development by maternal diabetes, some of which effects persist after birth and may continue to influence lung development during the period of postnatal alveolar septal growth.     

In situ hybridization and immunogold localization of vascular endothelial growth factor receptor-2 on the pericytes of the chick chorioallantoic membrane

This paper describes the expression of VEGF and of VEGFR-2 in the vasculature of the chorioallantoic membrane (CAM) as revealed by in situ hybridization and immunoelectron microscopy. Results showed that VEGFR-2 is expressed in both the endothelial cells and the pericytes, while VEGF in the chorionic epithelial cells. VEGF may therefore be released to promote both angiogenesis, by initiating an angiogenic response by endothelial cells expressing VEGFR-2, and the recruitment of pericytes along the capillary wall, playing also a crucial role in maturation and stabilization of the CAM blood vessels.     

Angiopoietin-2 and rat brain capillary remodeling during adaptation and deadaptation to prolonged mild hypoxia.

Angiogenesis is a crucial component of rat brain adaptation to prolonged hypoxia, but it is not known whether this structural change is permanent or reversed on return to normoxia. Also, the intrinsic mechanisms controlling brain microvascular plasticity in response to oxygen availability remains unclear. Our results indicate that capillary density in the rat cerebral cortex increased by 60% after 3 wk of hypoxia and that it progressively decreased to prehypoxic values after 3 wk of normoxic recovery (deadaptation). Angiopoietin-2 (Ang2) expression in the capillary endothelium was induced between 6 h and 14 days of hypoxia but fell to control levels at 21 days of hypoxia. During deadaptation, Ang2 levels were elevated at 1-14 days but decreased to baseline at 21 days. In contrast, the constitutive expression of Ang1 and Tie2 was not affected during hypoxia or deadaptation. TUNEL-positive endothelial cells and caspase-3 activation were observed at 7 and 14 days of deadaptation. These data suggest that Ang2 might modulate both angiogenesis and vascular regression in the rat brain and that capillary regression occurring during deadaptation involves activation of apoptosis.     

Developmental peculiarities of gas transporting system in chick embryo under respiratory surface restriction

The half of the chick eggshell surface was covered by the gas-proof layer on the 11th day and the incubation was continued up to the 14th day. The histological investigation of the chorioallantoic membrane after three days of experiment revealed that the blood vessels reduction in the covered part was completely compensated for by vessel dilatation and the growing in the mesodermal layer of intact part with parallel reorganization and dilation of capillary network on its surface. The blood gas capacity increase was supported for by haematocrit and haemoglobin growth and erythropoiesis stimulation. Cardiac work intensification was provided with the left ventricular myocyte hyperplasia and accelerated differentiation. The coordinated mobilization of all branches of gas transporting system compensated for surface half diminution to maintain the normal metabolic level in embryo during 11-14th days of incubation.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.