Chick Embryo Aorta Research Articles

Disruption of the aortic wall by coelomic lining-derived mesenchymal cells accompanies the onset of aortic hematopoiesis.

In vertebrates, definitive hematopoietic stem cells (HSCs) first emerge in the ventral wall of the aorta in the Aorta-Gonad-Mesonephros (AGM) region of the embryo, where they differentiate from a specialized type of endothelium termed Hemogenic Endothelium (HE). While the transition from HE to hematopoietic tissue has received much experimental attention, much less is known regarding generation of HE itself. The current study investigates the emergence of the HE in the chick embryo aorta. Using the HE marker Runx1 as well as a new chicken-reactive antibody to the endothelial marker VE-Cadherin, we document the relationship between the emerging HE and surrounding tissues, particularly the coelomic epithelium (CE) and CE-derived sub-aortic mesenchyme. In addition, the fate of the CE cells was traced by electroporation of a GFP-expressing plasmid into the CE, followed by analysis using immunofluorescence and in situ hybridization. We make the novel observation that CE-derived mesenchyme transiently invades through the ventral wall of the aorta during the period of establishment of HE and just prior to the emergence of hematopoietic cell clusters in the ventral aortic wall. These observations emphasize a hitherto unappreciated dynamism in the aortic wall during the period of HE generation, and open the door to future studies regarding the role of invasive CE-derived cells during aortic hematopoiesis.

Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis

We report for the first time that saliva of the hard tick and Lyme disease vector, Ixodes scapularis, is a potent inhibitor of angiogenesis. Saliva (< or = 1:500 dilutions) or salivary gland (0.1-0.5 pairs/assay) dose-dependently inhibits microvascular endothelial cell (MVEC) proliferation. Inhibition was also detected with the saliva of the cattle tick Boophilus microplus but not with the salivary gland of Anopheles gambiae, An. stephensi, Lutzomyia longipalpis, Phlebotomus papatasi, Aedes aegypti, Culex quinquefasciatus, and Cimex lectularius. Inhibition of MVEC proliferation by I. Scapularis saliva was accompanied by a change in cell shape (shrinkage of the cytoplasm with loss of cell-cell interactions) and apoptosis which was estimated by expression of phosphatidylserine using the Apopercentage dye, and by a typical pattern of chromatin margination, condensation, and fragmentation as revealed by nuclear staining with Hoechst 33258. The effect of saliva appears to be mediated by endothelial cell alpha5beta1 integrin, because monoclonal antibodies against this but not alphavbeta3, alphavbeta5, alpha9beta1, or alpha2beta1 integrins remarkably block its effect. In addition, SDS/PAGE shows that saliva specifically degrades purified alpha5beta1 but not alphavbeta5 or alphavbeta3 integrins. Incubation of saliva with EDTA and 1,10-phenanthroline, but not phenylmethylsulfonyl fluoride (PMSF), inhibits saliva-dependent degradation of purified alpha5beta1 integrin, suggesting that a metalloprotease is responsible for the activity. Finally, saliva at < or = 1:1,000 dilutions blocks sprouting formation from chick embryo aorta implanted in Matrigel, an in vitro model of angiogenesis. These findings introduce the concept that tick saliva is a negative modulator of angiogenesis-dependent wound healing and tissue repair, therefore allowing ticks to feed for days. Inhibition of angiogenesis was hitherto an unidentified biologic property of the saliva of any blood-sucking arthropod studied so far. Its presence in tick saliva may be regarded as an additional source of angiogenesis inhibitors with potential applications for the study of both vector and vascular biology.

Smooth Muscle Lineage Diversity in the Chick Embryo: Two Types of Aortic Smooth Muscle Cell Differ in Growth and Receptor-Mediated Transcriptional Responses to Transforming Growth Factor-β

Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if differences in primary embryonic lineage can give rise to SMCs with stable differences in growth and differentiation properties, we isolated Ect and Mes SMCs from the Day 14 chick embryo aorta. We report that despite different primary embryonic origins, Ect and Mes SMCs express nearly identical levels of seven SMC differentiation markersin vitro,consistent with their common smooth muscle developmental fatesin vivo.By contrast, Ect SMCs displayed a greater capacity for growth in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell–cell interactions. Most of the peptide growth factors tested that might account for serum-independent growth (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent differences in SMC responses to transforming growth factor-β (TGF-β). Exposure to TGF-β1 (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-β1 was growth inhibitory. In SMC cultures transfected with p3TP–lux, a luciferase reporter controlled by the TGF-β1-response elements of the human PAI-1 promoter, TGF-β1 (120 pM) produced 12 ± 2-fold increases in luciferase activity in Ect SMCs and only 3 ± 1.5-fold increases in Mes SMCs. Analysis of TGF-β receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-β receptors. However, using a polyclonal antibody specific for the chick type II TGF-β receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-β receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent differences in growth and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and growth modifiers, e.g., the TGF-βs. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to factors involved in the progression of vascular disease later in life.

Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter

DNase I footprinting experiments with a DNA fragment of the human elastin promoter have revealed a protected segment comprised between - 156 and - 172 nucleotides from the translation start site. Various types of gel retardation experiments indicate that the protected element binds different members of the C/EBP family of transcription factors. CAT (chloramphenicol acetyltransferase) fusion constructs carrying the wild type or a mutated promoter sequence were transfected into NIH3T3 and chick embryo aorta cells. The mutation significantly lowered CAT expression in NIH3T3 cells, but was ineffective in aorta cells. Cotransfection of the CAT promoter constructs with eucaryotic vectors expressing C/EBPs, did not affect the production of the reporter gene in NIH3T3 cells; on the contrary a several-fold increase of CAT activity was observed in aortic cells. This increase, however, was identical for the wild type and the mutated constructs. Taken together the data indicate that the elastin promoter contains a recognition site for proteins of the C/EBP family and that the function of this cis-acting element on basal elastin transcription varies with the cell type.

Identification of a TGF-β Responsive Element in the Human Elastin Promoter

In a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-β in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-β responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-β activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine.

Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).

Regulation of the human elastin promoter in chick embryo cells: Tissue-specific effect of TGF-β

The function of the 5′ flanking region of the human elastin gene on transcription regulation has been investigated in chick embryo aorta cells by transient DNA transfer experiments with elastin-chloramphenicol acetyltransferase (CAT) fusions. The results have shown that the region comprised within −129 and −12 bp from the translation start site is essential for transcription and probably contains different control sequences. Expression of the reporter gene was increased 2–4-fold by addition of TGF-β to the cell cultures. Analysis of CAT expression from different deletion constructs suggests that sequences in the region −196 to −12 play a major role in TGF-β induction. The stimulating effect of the growth factor could not be observed when transfections were performed with chick embryo tendon fibroblasts. This suggests that transcriptional regulation of elastin by TGF-β is tissue specific.

Biocompatibility of the Vascugraft ®: evaluation of a novel polyester methane vascular substitute by an organotypic culture technique

To evaluate the biocompatibility of chemically and structurally modified polyurethane elastomers for use as blood vessel replacements, small squares of vascular prostheses were cultured in direct contact with endothelium from chick embryo aorta using an organotypic culture assay. The polyurethane materials tested were: Vascugraft ® (fibrous, open pore structure); commercial Hydrophilic Mitrathane ® prosthesis (high porosity, smooth surface, non-permeable, closed pore structure); experimental hydrophobic Mitrathane (less porosity but a fibrous, open pore structure, similar to Vascugraft). The commercial expanded polytetrafluoroethylene prostheses Impra ® and reinforced GORETEX ® were included as controls on account of their extensive clinical application in the femoropopliteal position. After 5 d incubation at 37°C biocompatibility was assessed in terms of average area of migrating cells on the biomaterial, total number of cells surrounding the expiant and level of adhesion between the cells and the biomaterial. The Vascugraft prosthesis promoted the growth of a continuous monolayer of cells on its surface. This behaviour was equivalent to Impra and reinforced GORETEX materials in terms of cell density and area of cell migration but appeared to be superior for cell adhesion. From a second series of cell culture tests, in which the extractables leached from the biomaterials were added to the nutrient medium, it was concluded that none of the biomaterials tested released cytotoxic contaminants.

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.

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.

The wall of the chick embryo aorta harbours M-CFC, G-CFC, GM-CFC and BFU-E.

In the 3- to 4-day avian embryo, after the first wave of haemopoiesis which derives in the yolk sac from haemopoietic stem cells formed in situ, haemopoietic cells emerge in an intraembryonic site, the wall of the aorta. In this paper, we demonstrate that this site harbours M-CFC, G-CFC, GM-CFC and late and early BFU-E. In serum-free medium, the growth of M-CFC and GM-CFC was strictly dependent on CSF present in fibroblast-conditioned medium (FCM). The growth of G-CFC was improved when FCM was replaced by a minute quantity of chicken and fetal calf serum. Like erythroid progenitors from bone marrow, BFU-E detected here required anaemic chicken serum to differentiate into haemoglobinized cells. The frequency of the different types of haemopoietic progenitors in the aortic population was very high: 80 M-CFC, 25 G-CFC, 4 GM-CFC and 70 BFU-E for 12,500 aorta cells, i.e. two to eight times more frequent than in the bone marrow population, depending on the type of progenitors.

In vitro detection of cells with monocytic potentiality in the wall of the chick embryo aorta

Our previous investigations in 3- to 4-day avian chimeras have revealed that the wall of the aorta is a site from which hemopoietic stem cells can be obtained. In the present work using an in vitro clonal assay, we searched for cells with monocytic potentiality in this location as well as in the remainder of the embryo's body. In each experimental series thoracic segments from 30 chick embryo aortae were dissociated by a pancreatin treatment and plated in agar medium containing chicken serum and fibroblast-conditioned medium. Eighty to 620 macrophage colonies developed when 50,000 cells from 4-day aortae were plated, somewhat fewer when 3-day cells were plated (19–110). By contrast no progenitors were detected when cells were plated from 3- or 4-day embryos after their aorta had been removed. The cell composition and morphology of colonies deriving from aorta cells, their growth requirement and kinetics of development were identical to these of colonies deriving from young chicken bone marrow cells, cultured in the same conditions. The presence of macrophage progenitors in the wall of the 3- or 4-day embryo aorta and their absence in the rest of the embryo argues for a specific role of that region in embryonic hemopoiesis, namely that this is the location where intraembryonic hemopoietic stem cells emerge from the mesoderm at that period of development.

In vitro translation of elastin mRNA and processing of the translated products and the signal sequence of elastin a.

Chick embryo aorta mRNAs were translated in a gel-filtered reticulocyte lysate. The translated products showed two elastin-related proteins (a and b; relative mass approximately 70 000). The translated elastin a protein was separated essentially free of the b protein by centrifugation and sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. The a protein was then electroeluted from the SDS-polyacrylamide gel and a partial sequence was determined by automated Edman degradation. The NH2-terminal presequence of the elastin a protein is (formula; see text) The assigned sequence is identical to that reported for the b protein. Translation of chick embryo aorta mRNA in the presence of dog pancreas microsomal membranes segregated the a and b proteins into membrane vesicles and cotranslationally cleaved their respective presequences. The processed a and b elastin proteins were isolated together and NH2-terminal proline positions were determined. These are (formula; see text) These proline positions 4 and 8 are identical to those for NH2-terminal sequence for tropoelastin. This suggests that the signal peptidase removes the 24-residue signal peptide and thus directly generates the tropoelastin sequence, with no NH2-terminal prosequence as the intermediate.

Control of elastin synthesis.

mRNA was isolated from the thoracic aortas of 16-day chick embryos and used to synthesize blunt-ended heteroduplex molecules consisting of one strand of mRNA and one of cDNA using AMV reverse transcriptase and S1 nuclease. The duplexes were tailed with dCTP and hybridized to the plasmid pBR322 which had been restricted with Pst I and tailed with dGTP. Recombinant plasmids were used to transform E. coli C600 and colonies containing elastin cDNA were selected by in situ hybridization with 32P labeled elastin mRNA and by hybrid selected translation using the nuclease-treated reticulocyte lysate system. mRNA recovered from hybridization to DNA of one clone, pWB1, markedly stimulated incorporation of [3H]valine into a protein which was immunoprecipitable with elastin-specific antibody and had a molecular weight of 72,000, characteristic of tropoelastin. The 230 bp insert of pWB1 was sequenced by the technique of Maxam and Gilbert and found to be derived from a nontranslated region of the 3' end of the mRNA. Nick-translated pWB1 was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single mRNA species of 3.5 kb hybridized to the pWB1 probe and this species increased greatly in amount between day 7 and day 14. This increase was paralleled by an increase in translatable elastin mRNA and by the rate of elastin synthesis of aortas from various age embryos incubated in vivo. The injection of 150 microgram of hydrocortisone 21-phosphate into 8-day eggs produced a significant increase in both the relative rate of tropoelastin synthesized by the isolated aortas and the relative amount of elastin mRNA. These results suggest that the observed changes in elastin synthesis during development and after hydrocortisone administration are governed by the elastin mRNA content of the aortas.

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.

35] Lysyl oxidase: Preparation and role in elastin biosynthesis

This chapter describes purification and assay methods utilized for the study of aortic lysyl oxidase and its activity toward elastin substrates. Lysyl oxidase is mostly insoluble in neutral salt buffers in a variety of connective tissues, although enzyme activity is present in the growth medium of fibroblast cell cultures as a soluble protein. The enzyme can be solubilized from connective tissue sources by buffers containing 4–6 M urea. Most purification schemes that have evolved begin with the ureasolubilized fraction and generally share other purification steps as well. There are two assay methods for lysyl oxidase: tritium release assay and enzyme-coupled spectrophotometric assays. In tritium release assay chick embryo aortas are incubated in organ culture with [6- 3 H]lysine in the presence of BAPN. The salineinsoluble, elastin-rich fraction is isolated from the pulsed aortas after incubation in organ culture. Oxidative deamination of the E-carbon of radioactive peptidyl lysine in this insoluble substrate releases a tritium ion that forms tritiated water by exchange during the assay. Tritiated water is distilled in vacuo from assay mixtures and quantified by liquid scintillation spectrometry. In enzyme-coupled spectrophotometric assays the production of n-butyraldehyde by lysyl oxidase is coupled to the reduction of NAD + by aldehyde dehydrogenase, measuring the change in absorption at 340 nm.

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.

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.

Synthesis of an elastin component of molecular weight about 70,000 by polysomes from chick embryo aortas

Polysomes were isolated from aortas of 17-day-old chick embryos, and the synthesis of the nascent polypeptide chains was completed in vitro. When a mixture of a labeled amino acids found in elastin was used, the major radioactive product obtained was of molecular weight about 70,000 and was similar to elastin by several criteria. The 70,000 molecular weight product was extractable in propanol-butanol, it was not labeled with [ 35S]methionine, and it was precipitated by antibodies against elastin. Polypeptides larger than 70,000 molecular weight were also synthesized but these larger polypeptides incorporated relatively small amounts of [ 14C]valine, and they appeared to represent proα chains of procollagen. The results suggest that the major gene product for elastin has a molecular weight of about 70,000.

Synthesis of Elastin. A Rapid Formation of Lysine-derived Crosslinks by Chick Embryo Aorta

Aortas of 13-day-old chick embryo were labeled for 0.5 hr with [14C]lysine and subjected to a serial extraction after chase for 1-24 hr with [12C]lysine. Substantial radioactivity was found in insoluble elastin after 3 hr chase. The effect of beta-amino-propionitrile on labeling with [14C]lysine was also examined. Each fraction was hydrolyzed and applied to a short column on an amino acid analyzer. Radioactivity was found in desmosine and isodesmosine of insoluble elastin as early as 1 hr after the beginning of chase. The radioactivity increased rapidly at 2 hr and very slowly thereafter. A large count, which was separated into five peaks on a long column, was observed in other lysine derivatives at 2 hr and increased steadily up to 24 hr, while the lysine count decreased from 1 : 0.5 to 1 : 6 against lysine derivatives and from 1 : 0.04 to 1 : 0.9 against quarter-desmosine after 24 hr. The oxidation of lysine residues incorporated during the 0.5 hr pulse was almost completed during the first 1 hr of chase, and these oxidized residues were incorporated into crosslinks during the following 1 hr. It is suggested that poorly crosslinked elastin accumulated in the soluble fractions. The presence of crosslinking derived from lysine residues was also indicated in the microfibril fraction.