Chick Aorta Research Articles

Banded fibers in Tropoelastin coacervates at physiological temperatures

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

性ホルモンと動脈硬化症

Two trials of teststoerone feeding were preformed in order to test the effects of testosterone on serum lipid and aortas of chicks. In the first experiment, chicks were fed ad-libitum a diet which contained either 1% cholesterol or 0.1% testosterone or both for eight weeks. Chicks fed cholesterol only developed hypercholesterolemia and small lipid deposits in the interlammelar connective tissue cells of the aorta. There were no significant changes in the serum lipid level of testosterone supplemented chicks. Chicks fed testosterone adlibitum exhibited a mild degeneration of smooth muscle cells. In the second experiment, chicks were force-fed 150mg of testosterone alone per day for 7 weeks. Macroscopically, these chicks developed a precocious puberty-like appearance, which was characterized by a big comb and a short stature. Hypertriglyceridemia was also seen. Ultrastractural changes were marked in both thoracic and abdominal aortas. These changes consisted of lipid deposition, activation and degeneration in both interlammelar connective tissue cells and smooth muscle cells of entire aorta.

Analysis of elastin gene expression in the developing chick aorta using cloned elastin cDNA.

A segment of chick elastin cDNA cloned in the plasmid pBR322 was sequenced by the method of Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564) and the 192-base pair insert was found to be derived from the nontranslated region of the 3' end of the mRNA. The nick-translated cDNA was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single species of 3.5 kilobases hybridized to the cDNA probe and this species increased greatly between day 7 and day 14 of embryonic development. This increase was paralleled by an increase in translatable aortic elastin mRNA and by the in vivo rate of elastin synthesis, demonstrating that the change in synthesis during development is largely controlled by the elastin mRNA content of the aorta.

性ホルモンと動脈硬化症

In order to test the hypothesis that estrogen protect coronary arteriosclerosis, a systemic ultrastructural survey was conducted on arterial lesions in chicks fed various combination of cholesterol and estradiol. The combination of cholesterol and estradiol induced more hyperlipidemia than cholesterol alone. Lipid vacuoles were observed more frequently in the interlameller connective tissue cells than in the smooth muscle cells of the thoracic aorta of 1% cholesterol fed chicks. Estradiol supplementation with or without cholesterol caused lipid deposition in medial smooth muscle cells as well as in interlamellar connective tissue cells of the thoracic aorta. Moreover, both non-foam and foam cell type smooth muscle cell degeneration was observed in the abdominal aorta and coronary artery of chicks fed estradiol supplemented diets. These results suggested that estradiol has a potent angiotoxic effect.

36] Isolation of soluble elastin from copper-deficient chick aorta

Lysyl oxidase activity and the rate of cross-linking must be reduced effectively to cause an increase in the net pool of soluble elastins prior to isolation. This may be achieved by rendering animals nutritionally copper-deficient or lathrytic. The choice of experimental animals is also important. In this regard the chick system has been of fundamental importance because the isolation of soluble elastin from other common experimental animals, such as rats and mice, has proved to be difficult. If a nutritional or pharmacological treatment is used to precondition animals for subsequent isolation of soluble elastin, it is often necessary to initiate the treatment during periods of neonatal development. The chick is easily manipulated nutritionally during neonatal development, and with a sufficient number of chicks the yield of thoracic aorta, an excellent source of soluble elastin, is adequate for soluble elastin isolation.

Kinetics of the incorporation of tropoelastin into elastic fibers in embryonic chick aorta.

Matrix-free cells isolated by enzymic digestion of chick embryo aortas were labeled with [14C]proline for 20 to 60 min and the kinetics of the secretion of tropoelastin were followed by chasing the label and assaying [14C]tropoelastin in the cells and in the medium. The results indicated that secretion of tropoelastin followed the kinetics of a single first order process with a half time of 60 min. In parallel experiments tissue explants of chick embryo aortas were labeled with [14C]proline and the kinetics for the incorporation of tropoelastin into elastic fibers were followed by chasing the label and assaying the soluble [14C]tropoelastin and insoluble [14C]elastin in tissues. It was found that the incorporation of tropoelastin into elastic fibers also followed a single first order process with a half time of 85 min, similar to the secretion of tropoelastin from cells. In further studies, antibodies directed against tropoelastin were utilized to isolate soluble [14C]elastin components in the tissues after 0 to 4 hr chase of the 14C label. The results demonstrated that all soluble elastin components were recovered as monomeric tropoelastin and no soluble oligomeric elastin could be detected. These results are consistent with the proposition that elastic fiber growth occurs by addition of individual tropoelastin molecules to existing fibers and that oligomers of elastin were not intermediates in the process.

Differential expression of aortic and lung elastin genes during chick embryogenesis

The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.

Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E 1 and E 2, but not of prostacyclin and prostaglandin D 2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE 1 and PGE 2 could abolish all types of aggregation studied, whereas prostacyclin (PGI 2) and PGD 2 were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE 1 and PGE 2 inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently. The lack of any hemostatic function of PGI 2 in chickens was also indicated by the absence of biosynthesis of endogenous PGI 2 in chicken aorta. PGI 2 was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.

Lack of prostacyclin biosynthesis by aortic tissue of the chicken

The capacity of chicken aorta to produce prostaglandins both from exogenous and endogenous arachidonic acid has been evaluated. The metabolism of arachidonic acid in this tissue is mainly directed to PGE 2 and, in contrast to mammalian species, virtually no prostacyclin synthetase in present. However, the capacity of chicken thrombocytes to generate thromboxane A 2 and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid is similar to that observed for the mammalian blood platelets.

Copper deficiency and elastin metabolism in avian lung.

AbstractCopper deficiency in the chick causes anatomical changes in lung that are characterized by an apparent thinning of the air-blood capillary network of tertiary bronchi. Although the net content of the elastin in lung was not changed significantly by nutritional copper deficiency, it was possible to demonstrate an increase in the lysine content and a decrease in the desmosine content of lung elastin from copper-deficient chicks compared to that from controls. Further, the content of soluble elastin was also increased two- to fourfold in lung from copper-deficient chicks. Subsequently, nutritional copper deficiency was used to facilitate the isolation of chick lung tropoelastin. This lung protein appeared to be similar in selected properties to tropoelastin isolated from aorta of copper-deficient chicks. In addition, data are presented that indicate elastin in avian lung is possibly subject to only limited turnover.

Immuno electron microscopic studies on cells synthesizing elastin.

Embryonic chick and hamster aortas were examined in the electron microscope using ferritin-conjugated, elastin-specific antibodies after etching of the plastic, thin sections with a dilute solution of benzene, methanol and ethanol. Specific staining of intracellular vesicles was observed in both smooth muscle and endothelial cells in addition to extracellular elastin fibers. In the chick cells, these ferritin-stained vesicles had a lipid-laden appearance. In both species the vesicles appeared to fuse with the plasma membrane and discharge their contents into the extracellular space suggesting elastin is secreted via vesicular structures.

Identification of elastin in developing aortic tissue by immunological methods.

Antibodies raised in rabbits to tropoelastin isolated from lathyritic chick aorta were conjugated with ferritin and used to identify and locate elastin-containing elements in sections of aortic tissue from developing chicks. The ferritin-antibody conjugate was associated with a network of fine filaments with a diameter of 3-5 nm which, in the 8-10 day old chick embryo aorta, was distributed between and around small but recognizable elastic fibers. In older aortic tissue this ferritin-labelled material was seen principally in loose association with the periphery of developing elastic fibers. The microfibrils did not interact with the ferritin-antibody conjugate. Treatment of chicks with beta aminopropionitrile did not interfere with the accumulation of ferritin-labelled filaments on these small elastic fibers, suggesting that the mechanism of addition of new material onto growing fibers may be independent of cross-linking.

Optimal conditions for cell-free synthesis of elastin

Optimal conditions for the translation of elastin mRNA1 in a mRNA-dependent rabbit reticulocyte lysate were determined. Using total RNA isolated from embryonic chick aortae as the source of exogenous RNA, the concentrations of various components present in the translation assay were varied and the effect on elastin synthesis quantitated by immunoprecipitation. Components examined included: magnesium acetate, potassium chloride, spermidine, creatine phosphate, ATP, and GTP. In addition, it was found that heating of the RNA prior to translation significantly enhanced total protein synthesis, elastin synthesis, and the synthesis of proteins possessing molecular weights of greater than 80,000.

Correlation of functional elastin messenger ribonucleic acid levels and rate of elastin synthesis in the developing chick aorta.

Correlation of functional elastin messenger ribonucleic acid levels and rate of elastin synthesis in the developing chick aorta.

Characterization of molecular motions in 13C-labeled aortic elastin by 13C-1H magnetic double resonance.

AbstractPurified insoluble elastin samples labeled with [1‐13C]valine, [1‐13C]alanine, and [1‐13C]‐lysine were prepared from chick aorta in culture. The molecular mobility at the labeled sites was investigated using 13C‐1H magnetic double‐resonance spectroscopy. Linewidths, T1, and nuclear Overhauser effect (NOE) values of the labeled carbons alone were obtained from dipolar decoupled difference spectra. Analysis of these parameters together with signal intensity measurements showed that essentially all the valyl residues, ca. 75% of the alanyl residues, and ca. 60% of the lysyl residues were characterized by rapid backbone motions having τ = 65 nsec. Resonances due to the remaining alanyl and lysyl residues were detected in cross‐polarization experiments, which enhance the signals of motionally restricted carbons. Since lysyl and alanyl residues are found in the crosslink regions of elastin, whereas valyl residues are not, we conclude that crosslinks rather than secondary structures in the extensible region of the protein are the main source of motional restrictions in the protein. Elastin chain mobility was monitored by linewidth measurements over the range −90 to +70°C. When the swelling solvent (0.15M NaCl) was fixed at 0.6 g/g of elastin, a rapid monotonic reduction in chain mobility was observed as the temperature was lowered from 50 to 5°C. Liquidlike mobility was completely lost at 5°C. In contrast, the same sample in contact with excess solvent retained its liquidlike molecular mobility until −13°C, where it abruptly became rigid. The molecular mobility of this sample was temperature insensitive in the physiologically interesting range, 20–40°C, as a consequence of the opposing influences of temperature and swelling. Taken together these nmr data indicate that under physiological conditions, elastin is a network of mobile chains whose motions are strongly influenced by protein–solvent interactions.

High shortening velocity of isolated single arterial muscle cells.

Surprisingly high shortening velocities (less than 200 msec contraction-relaxation cycles) were found in isolated vascular muscle cells cultured from rat or chick aorta. All of the small fraction cells with such quick contractions had membrane excitation by short duration spikes, rather than the slower graded depolarization of the other cells which produced 20-fold slower contractions.

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [ 3H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M r ) and tropoelastin (70,000 M r ) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [ 3H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10 −8 m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.

The synthesis of soluble and insoluble elastin in chicken aorta as a function of development and age. Effect of a high cholesterol diet.

The synthesis of soluble elastin, newly synthesized insoluble elastin and total accumulated insoluble elastin was measured in aortic tissue of chickens ranging in age from the 11-day embryo to the adult chicken. Synthesis of soluble elastin reached a maximum in the 1st week after hatching, then decreased rapidly with a second transient increase between 4 and 6 weeks and thereafter decreased continuously until synthesis could no longer be detected in the 35-week-old adult. A portion of this newly synthesized soluble elastin was insolubilized even during 1 h of incubation by a β-aminopropionitrile-inhibited mechanism. Total insoluble elastin accumulated rapidly in aortic tissue in the late embryonic stages and reached a plateau about 1 week after hatching, after which time the proportion of the protein in the tissue remained constant. Synthesis of soluble elastin and total insoluble elastin was also determined in aortic tissue of chickens raised on a diet containing 2% cholesterol for 14 weeks after hatching. This cholesterol-rich diet had an early, but transient effect on the synthesis of soluble elastin, shifting the age of maximal synthesis to 1 to 2 weeks after hatching. However, by 6 to 8 weeks on the diet there was no difference in soluble elastin synthesis between normal and cholesterol-fed groups. Although prolonged cholesterol feeding resulted in serum cholesterol levels 10 times normal, aortic tissue cholesterol levels 3 times normal and grossly visible atherosclerotic lesions, no reinitiation of soluble elastin synthesis or alteration in the quantity and character of insoluble aortic elastin could be detected.

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.

Isolation and translation of elastin mRNA from chick aorta

mRNA was isolated from 15-day chick embryo aortae by digestion of the tissue at 40° with Proteinase K in 1% sodium dodecyl sulfate followed by chromatography on oligo(dT)-cellulose columns. The mRNA was translated using a reticulocyte lysate system and the tropoelastin immunoprecipitated using a specific antiserum prepared against insoluble elastin. The precipitated product was characterized by polyacrylamide gel electrophoresis and a major peak corresponding to a molecular weight of approximately 70,000 daltons was observed. No higher molecular weight product was observed.