Immunolocalization of endocan during the endothelial-mesenchymal transition process.

Endocan is a dermatan sulfate proteoglycan (DSPG) that has been observed in the cytoplasm of endothelial cells of small and large vessels in lung, kidney, liver, colon, ovary and brain tumors. This DSPG has been implicated in the regulation of cellular activities such as adhesion, migration, and proliferation. Given the important roles played by endocan in such processes, we sought to determine whether this DSPG is present in the chicken embryo aortic wall in embryonic days 12 and 14, when intimal thickening and endothelial transformation are notorious. Immunolabeling of serial paraffin cross-sections revealed endocan immunoreactivity at the endothelium and some mesenchymal cells constituting the intimal thickening but not in the cells arranged in lamellar layers. We also investigated whether endocan was present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin when they were deprived of serum and stimulated with epidermal growth factor. Immunofluorescence determined that in the epidermal growth factor (EGF) condition where separating, detaching, and migrating cells were observed, endocan appeared organized in arrays typical of focal complexes in the leading edge of these cells. In serum-free medium condition in which the endothelial cells displayed a cobblestone appearance, endocan appeared mainly delineating the margin of many cells. This study demonstrates for the first time the presence of endocan during the aortic wall remodeling, and provides evidence that suggests a possible contribution of this DSPG in the endothelial-mesenchymal transition (EndoMT) process.

[Effect of the lactose induced diarrhea on macronutrients availability and immune function in well-nourished and undernourished rats]. / Effecto de la diarrea inducida con lactosa sobre la disponibilidad de los macronutrientes y la función inmune en ratas nutridas y desnutridas.

In this study we compared the availability of nutrients in a balanced diet offered to young well-nourished and undernourished Sprague Dawley rats, with and without diarrhea. Malnutrition was induced by restricting food intake (50%) in one half of the rats for 2 weeks and diarrhea was induced by including 45% lactose in the diet after malnutrition had been established. During the experiment which lasted 8 d the animals were kept on the same feeding protocol but one half of the nourished and one half of the undernourished received lactose to induce diarrhea. The results showed that the inclusion of lactose at 45% in the diet caused a severe diarrhea both in the nourished and undernourished rats. This diarrhea however, resulted in a reduction in food intake and growth only in the well-nourished rats. In the rats with diarrhea the apparent digestibility of the diet and of its macronutrients decreased compared with the animals without diarrhea but this reduction was less apparent in the undernourished rats. Similar results were obtained in relation to the retention of nitrogen and energy. In this case, diarrhea was associated with retentions which were lower than those seen in the rats without diarrhea but the undernourished rats with diarrhea retained more energy than the well-nourished rats with diarrhea. Malnutrition resulted in lower packed cell volume, leukocyte count and thymus weight but diarrhea in the malnourished rats did not cause a further reduction in these variables as it did in the well-nourished animals. In general, these results indicate that in well-nourished rats, diarrhea had a negative effect whereas in the undernourished group it did not. It appears that the undernourished rats compensated their nutrient utilization so that diarrhea did not worsen their undernourished condition.

Characterization of nonmuscle cells present in the intima of normal adult bovine pulmonary artery.

In this study, the presence of cells in the intimal region of normal adult bovine pulmonary artery (BPA) was examined by analysis of longitudinal sections at the level of light and transmission electron microscopy. In addition, the morphological and immunohistochemical phenotype of these cells as well as the presence of particular extracellular matrix (ECM) components in this region were also determined. Since ECM production and cell proliferation have been demonstrated to be regulated by locally released growth factors such as transforming growth factor beta (TGFbeta), the presence of TGFbeta-1 in this region was also investigated. Our findings reveal the presence of immature or "nonmuscle" cells into the subendothelial space of normal adult BPA. These cells were characterized by the presence of abundant cytoplasmic organelles and scanty microfilaments. Such cells were negative to antibodies against smooth muscle alpha actin (SM alpha-actin), 1E12, and vWf, but not to vimentin. Similar cells have recently been detected in normal adult BPA and canine carotid arteries, but in the medial region. Because of their location in these elastic arteries, the nonmuscle cells are involved not only in the remodeling of the medial region, but also in the neointima or intimal thickening formation by migration from the media to the subendothelial space, where they proliferate and secrete ECM components. However, a limited number of morphological studies and the current investigation describe the presence of scattered nonmuscle cells within the intima of some normal elastic arteries. This would suggest an important role for these resident cells within the intima in normal and pathological processes as well. In addition, our results show the presence, in this region, of TGFbeta-1 and of ECM components that include collagen, elastin, fibronectin, and laminin which are present in normal conditions and during the intima formation in vivo.

Intimal thickening involves transdifferentiation of embryonic endothelial cells.

Morphological studies have hypothesized different origins for the precursors of the vascular smooth muscle cells (SMCs). The intriguing possibility that intimal SMCs may arise from the endothelium has newly emerged. As a first step towards understanding of the possible mechanisms involved in the transdifferentiation of endothelium into smooth muscle cells, we characterized the in vivo phenotype of the cells located in the aortic wall (distal to the aortic arches). This was accomplished using advanced stages of chicken embryo development. Furthermore, we investigated whether the cells present at the intimal thickening derive from the endothelial cell transdifferentiation. Immunolabeling of serial cryosections suggested that mesenchymal cells observed in the intimal thickening may arise from the endothelium. These cells may persist either as non-muscle throughout the development or possibly convert to cells expressing smooth muscle alpha-actin (SM alpha-actin). To determine whether endothelial cells may actually transdifferentiate into mesenchymal cells, aortic explants from 14-day-old chicken embryos (stage 40) were used. We found that explanted endothelial cells lose their cobblestone-appearance and migrate toward cell-free area. Some of these cells maintain the vWf immunoreactivity, whereas other cells coordinately lose vWf and gain SM alpha-actin expression (transitional cells). Taken together these findings strongly support the possibility that embryonic aortic endothelial transdifferentiate into mesenchymal cells, some of which express SM alpha-actin. Since TGFbeta-3 is considered an essential factor during epithelial to mesenchymal transitions in earlier chicken heart development, we also investigated the distribution of this growth factor at day 14. Our observations indicated that the immunoreactivity for TGFbeta-3 in this stage may be associated with migrating mesenchymal cells and that this immunoreactivity appears to decrease as cell differentiation advances. Therefore, the present study provides evidence that could help to explain 1) the presence of cells displaying a phenotype reminiscent of fetal-like cells in the normal chicken aorta and in the intimal region of the human aorta; 2) the SM lineage diversity in the chicken embryo reported by others; 3) a subpopulation of immature cells in the subendothelial region of the main pulmonary arteries of fetal, neonatal and adult bovines; and 4) the presence of intimal cushions, intimal pads, eccentric and diffuse intimal thickening that are observed in mammalian and avian vessels at birth.

[Serum lipids and vitamin A, C and E concentrations in an adult population of Caracas City]. / Lípidos séricos y niveles de las vitaminas A, C y E en una población adulta de la ciudad de Caracas.

Lipid profiles as well as vitamins A, C and E were determined in a sample of 90 men and 151 women with ages within 35 and 50 years old. Comparing the lipid profiles obtained in the study with the limits established by the National Cholesterol Education Program, resulted that a little more than 60% of the group had total cholesterol and LDL cholesterol levels in the desirable range, 20 to 30% had levels in the marginal range while 10 to 15% had levels in the high risk range. This distribution of the risk is more favorable than that observed in populations with a high risk of heart diseases such us the British or American populations, which show a substantially higher segment of the people in the high risk level. When the protective effect of the HDL cholesterol was included in the estimation of the risk by calculating the indexes: Total cholesterol/HDL chol., LDL Chol/HDL chol. or Total Chol-HDL chol. 65 to 80% of the population had values within the normal range and the first of these indexes, indicated that the men had a higher risk than the women. Integrating both methods of estimating the risk and considering that the risk of the individuals in the marginal range defined by the National Cholesterol Education Program is minimal unless they have two additional risk factors, it appears that an important segment (20-30%) of the studied population may benefit from programs aiming to reduce other risk factors such as smoking, high blood pressure, diabetes or overweight. The vitamin levels measured in this study indicated that the vast majority of the population had their levels in the safe range but an important segment had vitamin C serum levels indicative of poor consumption of this vitamin. Since vitamin C is high in fruits and vegetables we concluded that the studied population had a low consumption of these foods. Due to the existing evidence of a protective effect of fruits and vegetables in heart and other chronic diseases it was concluded that institutions such as the one studied here should engage in preventive campaigns emphasizing a reduction of both risk factors and the consumption of saturated fats. The results of this study also indicate that the consumption of fruits and vegetables should be encouraged.

Characterization of the atherosclerotic plaque in the internal mammary artery.

The current study examines, at both structural and ultrastructural levels, representative segments of internal mammary arteries obtained from 15 male patients, ranging in age from 45 to 75 years, with signs or symptoms of coronary heart disease. These segments were obtained at the time of coronary bypass surgery. Of the 15 segments examined, only 2 were found to have atherosclerotic plaques. In other segments, only an intimal thickening similar to that observed during aging was found. There was evidence of endothelial cell loss and defects of internal elastic lamina in the present study; however, there was no evidence of lipid accumulation in the intimal region. This observation agrees with previous findings that indicate that lipid accumulation is not a necessary factor for the formation of atherosclerotic plaques. During the study microfilament bundles, the so-called "stress fibers," were also observed in the cytoplasm of the luminal side of endothelial cells. Stress fibers are known to be present in some endothelial cells in some pathologies such as regeneration after injury or hypertension. One of the features of the atherosclerotic plaques from an internal mammary artery was the presence of cells with contractile and synthetic phenotypes (contractile and synthetic smooth muscle cells), as well as cells with intermediate features. Cells with similar characteristics have also been observed during the development of the early stages of atherosclerosis, during embryological development of vessels, after experimental excimer laser treatment, and in primary cell culture. To our knowledge, this is the first report showing the ultrastructural features of the atherosclerotic plaques in the internal mammary artery.

Pericyte differentiation.

Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo.

Changes in plasma fibronectin in children after elective repair of congenital heart defects.

Plasma fibronectin is an attachment protein important for maintaining capillary integrity and host defense mechanisms. Depletion of plasma fibronectin has been shown to occur in adults after septic shock, major trauma, and burns. Limited laboratory and clinical studies suggest a correlation between decreased plasma fibronectin levels and increased pulmonary capillary permeability and tissue perfusion. Mild and transient plasma fibronectin depletion has been observed in adults after cardiovascular operations. We measured plasma fibronectin by immunoturbidometric assay in 20 children (age 6 months to 12 years) undergoing repair of congenital heart defects. Plasma fibronectin levels immediately after operations and daily thereafter were compared with the preoperative values. Plasma fibronectin declined on postoperative days 1, 2, 3, 4, and 5 (p < 0.05). A nadir was reached on day 3 with a tendency toward recovery thereafter. Patients with a therapeutic intervention score of more than 35 had greater magnitude of plasma fibronectin decline than those with a score of less than 35 at 24 hours after the operation (p < 0.005). We conclude that (1) significant and prolonged plasma fibronectin depletion occurs after cardiovascular operations in children; and (2) postoperative plasma fibronectin depletion is associated with increasingly complex surgical intervention. Reduced plasma fibronectin synthesis and more extensive operations for congenital heart defects are likely reasons for children being more susceptible than adults to plasma fibronectin depletion after cardiovascular operations.

Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.

Alpha-smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of alpha-smooth muscle actin (alpha-SM actin) may be induced to express the latter when exposed to TGF-beta 1. FVIII was detected by immunofluorescence, alpha-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing alpha-SM actin increased with time of incubation with TGF-beta 1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and alpha-SM actin after 5 days of incubation with TGF-beta 1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed alpha-SM actin. Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-beta 1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-beta 1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and alpha-SM actin-negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved chronic epicardial pacing in children: steroid contribution to porous platinized electrodes.

Although new "low threshold" epicardial electrodes combine steroid with a porous, platinized-platinum surface, the actual contribution of steroid elution has not been established. We evaluated this new electrode surface design with and without steroid in 13 children, ages 1-22 years. Both electrodes are unipolar and of similar surface area. The Medtronic Model 4951-P is a barb design for epimyocardial insertion without steroid while the Model 10295A is a steroid eluting, epicardial disk-shaped design. Both electrodes were implanted for atrial and ventricular pacing. At implant, sensed P and R waves, and pacing impedances were comparable between both electrodes. There were no significant differences between initial measured pulse width or calculated energy thresholds for the first 2 months following implant. Strength-duration curves for both electrodes at 1 month were comparable to implant values. After 2 months, the threshold of the nonsteroid electrode peaked and stabilized at a significantly higher (P less than 0.05) level than the more constant steroid eluting electrode. This difference continued for the first year following implant. We conclude that the new porous, platinized-platinum electrode design intrinsically limits initial electrode-tissue interface reactivity in children and improves epicardial pacing with low chronic threshold values. Steroid elution augments these intrinsic qualities by maintaining fibrous capsule stability with more constant low thresholds over time.

Concomitant changes in endothelial cell junctions and extracellular matrix components in the chick embryo aorta.

According to previous studies, a process of endothelial activation seems to be occurring in the chick embryo between days 7 and 18. Also, endothelial cells respond to collagen as a substratum between 12 and 18 days, and this response diminishes until it almost disappears after birth. In the present study, aortas from chick embryos (days 7 to 21), and from chicks (14 days posthatching) were used. The results obtained by the freeze-fracturing technique, showed that between days 12 and 14 the intramembranous particles were aggregated into linear or clustered arrays in the fracture P-face of endothelial cells. This could signify that some kind of gap junction-like coupling may occur between adjacent endothelial cells. Our results also indicate that in advanced stages (21-day-old chick embryos and 14-day-old chicks) the growth of small aggregates into larger aggregates or plaques could occur. In addition to gap junctions, the presence of macular and linear tight junctions, reported as focal tight junctions (day 14 of development) macular and linear tight junctions with free-ending strands oriented parallel to one another (21 days) and smooth contoured ridges (14 days post-hatching) were observed. This sequence of changes may represent a development from linear to macular, to a more occluding arrangement, and may also reflect an endothelial cell polarization. Histochemical study of proteoglycans was done by using cuprolinic blue according to the critical electrolyte concentration method. Cuprolinic blue-positive granular, elongated and microfibrillar materials were found in the subendothelial region, forming a meshwork that occupies the extracellular space. Qualitative and quantitative changes were observed both in proteoglycans and in other extracellular matrix components throughout development, suggesting an increase in extracellular matrix complexity. These results lead us to suggest that the assembly of a more complex extracellular matrix, concomitantly with the formation of intercellular junctions during development, might influence the polarization of endothelium in the aorta of the chick embryo.

Extracorporeal membrane oxygenation for the circulatory support of children after repair of congenital heart disease.

We have treated 39 infants and children with congenital heart disease with extracorporeal membrane oxygenation during the past 5 years. Thirty-six were treated for low cardiac output or pulmonary vasoreactive crisis after repair of congenital heart defects. Twenty-two (61%) survived. Most patients were cannulated from the neck via the right internal jugular vein and the right common carotid artery. Six patients were cannulated from the chest, including three who had separate drainage of the left side of the heart with a left atrial cannula. Two of these patients survived and were the only survivors of the nine patients cannulated in the operating room because they could not be weaned from cardiopulmonary bypass after open cardiac operations. We also reviewed 312 patients (the predictor study series) having open cardiac operations before the availability of extracorporeal membrane oxygenation; 27 of these patients died. Data were collected at 1 and 8 hours postoperatively to determine if any parameters might predict early mortality. With these parameters used as criteria, patients who went on extracorporeal membrane oxygenation were as sick as those who died before extracorporeal membrane oxygenation was available. The most common complication was bleeding related to heparinization. The mean transfusion requirement in survivors was 1.50 +/- 1.13 ml/kg/hr, 5.63 +/- 7.0 ml/kg/hr in the nonsurvivors, and 7.46 +/- 8.29 ml/kg/hr in those cannulated in the operating room because they could not be weaned from bypass. Four children had intracranial hemorrhage, and two of them died. There was one late death. Nine of the 22 survivors are entirely normal. All survivors who do not have Down's syndrome are considered to have normal central nervous system function. We conclude that extracorporeal membrane oxygenation can improve survival in patients with both pulmonary artery hypertension and low cardiac output after operations for congenital heart disease.

Down's syndrome, complete atrioventricular canal, and pulmonary vascular obstructive disease.

We reviewed our experience over a 10-year period to determine whether children with Down's syndrome and complete atrioventricular canal develop pulmonary vascular obstructive disease earlier than children with normal chromosomes and this defect. Comparisons were made between Down's syndrome and normal chromosome children regarding (1) pulmonary blood flow and pulmonary vascular resistance at initial catheterization, (2) operability as related to elevation in pulmonary vascular resistance, and (3) age at diagnosis of fixed pulmonary vascular obstructive disease. The 45 patients with Down's syndrome catheterized under 1 year of age had a lower mean pulmonary blood flow (3.2 versus 5.7; p = 0.0001) and higher mean pulmonary vascular resistance (8.3 versus 4.6 Wood units.m2; p = 0.0003) than their 34 normal chromosome counterparts. When all ages were included, 38 of 81 (47%) of the children with Down's syndrome and 32 of 40 (80%) of the normal children were considered operable. Non-Down's syndrome patients who had operations had a higher pulmonary blood flow (5.8 versus 3.3; p = 0.004) and lower pulmonary vascular resistance (3.6 versus 6.0 Wood units.m2; p = 0.005) than Down's syndrome patients. Of the 34 patients who did not have operations because of pulmonary vascular obstructive disease, 31 had Down's syndrome. In 10 of 81 children with Down's syndrome, fixed pulmonary vascular obstructive disease was diagnosed before the age of 1 year, while this was found in none of 40 normal children. Our data demonstrate that Down's syndrome patients with complete atrioventricular canal have a greater degree of elevation of pulmonary vascular resistance in the first year of life and more rapid progression to fixed pulmonary vascular obstructive disease than children with normal chromosomes.

Exercise performance after repair of anomalous origin of the left coronary artery from the pulmonary artery.

Eleven patients underwent exercise testing after operative repair of anomalous origin of the left coronary artery from the pulmonary artery. Five patients repaired after 2 years of age comprised a childhood surgery group, and six patients repaired before 2 years of age comprised an infant surgery group. All patients were exercised using either a treadmill or electronically braked bicycle with simultaneous thallium 201 scintigraphy. Oxygen consumption, carbon dioxide production, pulmonary functions, and electrocardiogram were all monitored continuously. Pulmonary reserve was normal in all patients. Based on heart rate reserve, respiratory exchange ratio, and oxygen-consumption response to work load, two patients in the infant surgery group stopped exercise before achieving maximum aerobic capacity. All remaining patients achieved their maximum aerobic capacity. There was no difference in work rate or oxygen consumption during exercise between the infant and childhood surgical group. Four patients (two in each surgical group) had an impaired chronotropic response to exercise. Three of these four patients demonstrated perfusion defects by thallium scintigraphy. Thallium scintigraphy was normal in all remaining patients. Electrocardiographic abnormalities were noted in seven of 11 patients having ventricular arrhythmias or ST segment depression. It is concluded from this study that exercise performance after repair of anomalous origin of the left coronary artery from the pulmonary artery is not affected by the age at which surgery is performed. Exercise is frequently associated with electrocardiographic evidence of abnormal myocardial perfusion despite frequently negative simultaneous 201Tl scintigraphy.

Tetralogy of Fallot.

Tetralogy of Fallot is the most common malformation of children born with cyanotic heart disease, with an incidence of approximately 10 per cent of congenital heart disease. There can be a wide spectrum as to the severity of the anatomic defects, which include ventricular septal defect, aortic override, right ventricular outflow tract obstruction, and right ventricular hypertrophy. Cyanosis may vary from mild to severe, and patients may present as newborns or, more commonly, young infants. Infants with classic tetralogy of Fallot and stable anatomy should undergo primary complete intracardiac repair. The overall hospital mortality is approximately 3 to 5 per cent, with most patients who survive having an excellent clinical and hemodynamic result.

Ultrastructural localization of specific surface antigens in the dimorphic fungus Sporothrix schenckii.

The ultrastructural localization of antigens recognized by an antiserum raised against formaline-killed Sporothrix schenckii yeast cells was investigated on yeast and mycelial phases of the fungus. Immunogold procedures revealed that these antigens were located on the cell surface of both growth phases. Labelling was heterogeneous and involved areas of Concanavalin A-binding sugar residues.

Behaviour of chick embryo aortic cells obtained through nonenzymatic means cultured onto collagen gels.

In the present paper we used a method whereby some of the cellular events that take place in the aortic wall during chick embryo development can be studied in vitro. Collagen gels were utilized to culture endothelial cells obtained through nonenzymatic means from aortic explants isolated from 12- to 14-day-old chick embryos. These cells were characterized by morphological and immunocytochemical criteria. After 72 h, explanted endothelial cells from embryonic aorta formed a monolayer of polygonal cells, which gave rise to elongated cells that migrate into the collagen gel. These cells are similar to those of mesenchyme-like cells observed in vivo at the subendothelial region of 14-day-old chick embryonic aorta. In long-term cultures, some of these cells acquired features either of synthetic smooth muscle cell phenotype, or of fibroblast-like cells very similar to those found in developing aorta. These results indicate that the culture of explants of chick embryo aorta on three-dimensional collagen gel is a valuable system for studying some of the complex morphogenetic events that occur during the development of the aorta.

Development of the aorta in the chick embryo: structural and ultrastructural study.

A structural and ultrastructural study was designed to analyze systematically the cellular events which take place in the aortic wall between days 7 and 21 of chick embryo development. Between days 7 and 18, increase in total diameter, number of cell layers, and aortic wall thickness are highly correlated, whereas between days 18 and 21 the total diameter increase is correlated mainly with an increase in vessel lumen diameter. Cell layers of smooth muscle cells showing an immature or synthetic phenotype arise from progressive association and organization of mesenchymal cells originated from an endothelial activation process in which a hyaluronic acid-rich extracellular matrix seems to be involved. It is suggested that the process of endothelial activation takes place between days 7 and 18 of embryonic development provided that within that period the typical cellular events which are involved in such a process take place (hypertrophy, reorientation, invagination, mitotic activity, acquisition of migratory appendages, endothelial detachment and incorporation into adjacent spaces). This endothelial activation has been recognized as a selective multiphasic process required for the transition of endothelial cells into mesenchyma.