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PubMed1987

Identification of multiple tropoelastins secreted by bovine cells.

High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.

Amino Acids↗

PubMed2002

Gene expression of tropoelastin is enhanced in the aorta proximal to the coarctation in rabbits.

To assess elastin biosynthesis in the aortic wall in response to acute elevation of blood pressure, we studied the aortic gene expression of tropoelastin in a rabbit midthoracic aortic coarctation model. The time points of the study were 1, 3, and 7 days and 2, 4, and 8 weeks after coarctation. Additional animals were subjected to hypercholesterolemia for analysis of tropoelastin expression in the intimal lesion. mRNA for tropoelastin was quantitated by Northern blot analysis and its distribution was revealed by in situ hybridization. The 65-kDa tropoelastin was analyzed by Western blotting and immunohistochemistry. Tropoelastin mRNA proximal to the coarctation was increased at 2 weeks and returned to baseline by 8 weeks (P < 0.05 versus control). Changes in 65-kDa tropoelastin corresponded to those of mRNA. Tropoelastin gene was expressed mainly in the intima and in the outer media at the proximal region to the stenoses, which was particularly remarkable in the intimal lesion. The results indicate that tropoelastin gene expression was enhanced in the early remodeling response to elevated blood pressure. The distribution of newly synthesized tropoelastin in the outer media suggests a reenforcement role of tropoelastin, which preserves mechanical resiliency in response to changes in tensile stress.

Animals↗

PubMed1989

Characterization of rat heart tropoelastin.

Several overlapping rat tropoelastin cDNA clones were isolated from a lambda gt11 rat heart cDNA library and their nucleotide sequence was determined. The corresponding deduced amino acid sequence of rat tropoelastin revealed strong homology to bovine and human tropoelastins although possessing some unique features including greater size (18%) and composition of repetitive units. Comparison of the amino acid sequence of rat tropoelastin to four other tropoelastin species reveals that the hydrophobic peptide repeat regions in the middle of each molecule and the crosslinking areas containing three lysine residues are remarkably conserved. A possible function for the clustering of three lysine residues in providing a mechanism for the in vivo reduction of dehydrolysinonorleucine via a redox shuttle with dihydrodesmosine is proposed. In addition, the COOH-terminal sequence of the rat tropoelastin is virtually identical to tropoelastins of other species in possessing a cysteine/arginine/lysine containing segment. There are no obvious amino acid insertions or substitutions in the COOH-terminal half of the rat tropoelastin molecule which would signal unique cleavage or glycosylation sites. Examination of the steady-state levels of rat tropoelastin mRNA in 8- and 12-day neonatal lung, heart, and aortic tissues showed that the amount of tropoelastin mRNA was abundant and of similar size (3.9 kb) in all three tissues.

Amino Acid Sequence↗

PubMed2006

Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins.

Tropoelastin is the monomeric form of elastin, a polymeric extracellular matrix protein responsible for properties of extensibility and elastic recoil in connective tissues of most vertebrates. As an approach to investigate how sequence and structural characteristics of tropoelastin assist in polymeric assembly and account for the elastomeric properties of this polymer, and to better understand the evolutionary history of elastin, we have identified and characterized tropoelastins from frog (Xenopus tropicalis) and zebrafish (Danio rerio), comparing these to their mammalian and avian counterparts. Unlike other species, two tropoelastin genes were expressed in zebrafish. All tropoelastins shared a predominant and characteristic alternating domain arrangement, as well as the fundamental crosslinking sequence motifs. However, zebrafish and frog tropoelastins had several unusual characteristics, including increased exon numbers and protein molecular weights, and decreased hydropathies. For all tropoelastins there was evidence of evolutionary expansion of the proteins by extensive replication of a hydrophobic-crosslinking exon pair. This was particularly apparent for zebrafish and frog tropoelastin genes, where remnants of sequence similarity were also seen in introns flanking the replicated exon pair. While overall alignment of mammalian, avian, frog and zebrafish tropoelastin sequences was not possible because of sequence variability, the C-terminal exon was well-conserved in all species. In addition, good sequence alignment was possible for several exons just upstream of the putative region of replication, suggesting that these conserved domains may represent 'primordial' core sequences present in the ancestral sequence common to all tropoelastins and in some way essential to the structure/function of elastin.

Amino Acid Sequence↗

PubMed1992

Phorbol ester-mediated downregulation of tropoelastin expression is controlled by a posttranscriptional mechanism.

Expression of tropoelastin, the principal precursor of elastic fibers, is tissue-specific and is limited to a brief developmental period. Little is known, however, about the mechanisms that regulate the tissue- and temporal-specific expression of elastogenesis. The tropoelastin promoter contains putative phorbol ester responsive elements, or AP-1 binding sites, but the functional significance of these sequences is unknown. To test if tropoelastin expression is influenced by phorbol esters, we exposed elastogenic fetal bovine chondrocytes to 10(-7) M 12-O-tetradecanoylphorbol 13-acetate (TPA). Tropoelastin mRNA levels decreased greater than 10-fold in response to TPA, and this downregulation was paralleled by a decline in the secretion of tropoelastin protein into the culture medium. As determined by nuclear-runoff assay and transient transfection with a human gene promoter-CAT construct, tropoelastin transcription was unaffected after exposure to TPA. As indicated by actinomycin D experiments, the half-life of tropoelastin mRNA in control cells was about 20 h, but exposure to TPA resulted in an accelerated decay of the tropoelastin transcript (t1/2 = 2.2 h). These data indicate that downregulation of tropoelastin expression was controlled by a posttranscriptional mechanism and that the AP-1 elements in the bovine tropoelastin promoter may not be involved in regulation of production.

Animals↗

PubMed2002

Biphasic response of tropoelastin at the poststenotic dilation segment of the rabbit aorta.

OBJECTIVE: The purpose of this study was to assess the time course of tropoelastin gene expression in the poststenotic dilatation segment of rabbit aorta with experimental coarctation. METHODS: Midthoracic aortic coarctation was created in rabbits to produce a PSD. The time points of the study after coarctation were 1, 3, and 7 days and 2, 4, and 8 weeks (n = 3 each). Additional animals (n = 6) were subjected to hypercholesterolemia for analysis of tropoelastin expression in intimal lesions. Northern and Western blot analyses were used to quantitate tropoelastin messenger RNA (mRNA) and protein, and immunohistochemistry was used to analyze tropoelastin distribution. RESULTS: Thoracic aortic coarctation produced a moderate stenosis, which resulted in PSD. mRNA levels in the PSD segment decreased at days 1 and 3, followed by an increase at 2 and 4 weeks (P <.05 versus controls). This biphasic change in tropoelastin mRNA was associated with increase in tropoelastin protein levels at 2 and 4 weeks (P <.05 versus controls). PSD diameter reached a maximum at 4 weeks and did not increase significantly thereafter. The number of medial elastic laminae in PSD was reduced slightly, but media thickness was unchanged. Intimal lesions were much smaller in the PSD segment than in the proximal segment in animals with hypertension superimposed with hypercholesterolemia. Moreover, tropoelastin protein distributed not only in the intima but also in the media of the PSD. CONCLUSION: Tropoelastin gene expression is regulated in a biphasic pattern and precedes PSD formation. The differential distribution of tropoelastin in the media suggests a role for tropoelastin in the poststenotic adaptation response, which may provide increased elasticity to the PSD wall.

Animals↗

PubMed2006

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts.

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

Animals↗

PubMed1982

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.

Animals↗

PubMed1983

Monoclonal antibodies as probes for differentiating tropoelastin a and b.

BALB/C mice were immunized with a mixture of aortic tropoelastin a and b. To ensure the presence of tropoelastin a, the lathyritic chick aortic tissue was first extracted with denaturing and reducing agents. After dialysis, the standard procedure for tropoelastin purification was followed. Following immunization, the lymphocytes were fused with a myeloma cell line. Resulting hybridomas were cloned and tested for reactivity towards tropoelastin a and b. The majority of monoclonal antibodies exhibited crossreactivity between the two tropoelastins. Two of the clones were further characterized because of their ability to differentiate both tropoelastin a and b. As demonstrated by immunoprecipitation and Western blot analyses, one clone reacted preferentially with tropoelastin b and the other with tropoelastin a and b. Significantly, these results demonstrate that tropoelastin a and b can be differentiated by immunological techniques.

Animals↗

PubMed1999

Deficient coacervation of two forms of human tropoelastin associated with supravalvular aortic stenosis.

Human tropoelastin associates by coacervation and is subsequently cross-linked to make elastin. In Williams syndrome, defective elastin deposition is associated with hemizygous deletion of the tropoelastin gene in supravalvular aortic stenosis (SVAS). Remarkably, point-mutation forms of SVAS correspond to incomplete forms of tropoelastin which include in-frame termination by nonsense mutations, yet the resulting phenotype of these disorders is not explained because expression variably occurs from both normal and mutant alleles. Proteins corresponding to two truncated tropoelastin mutants were expressed and purified to homogeneity. Coacervation of these proteins occurred as expected with increasing temperature, but substantially contrasted with that of the performance of a normal tropoelastin. Significantly, association by coacervation of the truncated SVAS tropoelastin molecules was negligible at 37 degrees C, which contrasted with the substantial coacervation seen for normal tropoelastin. Furthermore their midpoints of coacervation increased and correlated with the extent of deletion, in accord with the loss of hydrophobic regions required for tropoelastin association. Their secondary structures are similar, as evidenced by CD studies. We propose a model for point-mutation SVAS in which aberrant tropoelastin molecules are incompetent and are mainly excluded from participation in coacervation and consequently in elastogenesis. These forms of SVAS may consequently be considered functionally similar to a hemizygous deletion, and mark point-mutation SVAS as a disorder of defective coacervation.

Amino Acid Substitution↗

PubMed1995

Developmental regulation of elastin production. Expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels.

To assess the mechanisms controlling the developmental regulation of tropoelastin expression in vivo, we developed a reverse-transcription-polymerase chain reaction (RT-PCR) assay to detect tropoelastin pre-mRNA as an indicator of ongoing transcription in intact tissue. RNA was isolated from mid-fetal (early-elastogenic), neonatal (peak tropoelastin expression), and adult (very low tropoelastin expression) rat lungs and reverse transcribed, and the cDNA was amplified with intron specific primers. A weak hybridization signal for tropoelastin pre-mRNA was seen in mid-fetal samples, and paralleling the increase in steady-state mRNA levels, a strong signal for pre-mRNA was detected in neonatal samples, indicating transcriptional regulation. Stimulation of fetal lung tropoelastin expression by maternal administration of dexamethasone also led to an increase in pre-mRNA levels. However, signal for tropoelastin pre-mRNA in adult samples was equal to that detected in neonatal samples, even though mRNA levels had dropped about 80-fold. Persistence of tropoelastin transcription in adult tissue was also seen in cell culture models and was verified by nuclear runoff assay. In addition, an RT-PCR assay for alpha 1 (I) procollagen pre-mRNA accurately revealed the known transcriptional regulation of this gene. Our results demonstrate that the induction and maintenance of elastogenesis is controlled by a transcriptional mechanism, whereas, the cessation of tropoelastin expression is controlled by a post-transcriptional mechanism.

Aging↗

PubMed1998

Identification of tropoelastin as a ligand for the 65-kD FK506-binding protein, FKBP65, in the secretory pathway.

The folding and trafficking of tropoelastin is thought to be mediated by intracellular chaperones, although the identity and role of any tropoelastin chaperone remain to be determined. To identify proteins that are associated with tropoelastin intracellularly, bifunctional chemical cross-linkers were used to covalently stabilize interactions between tropoelastin and associated proteins in the secretory pathway in intact fetal bovine auricular chondrocytes. Immunoprecipitation of tropoelastin from cell lysates after cross-linking and analysis by SDS-PAGE showed the presence of two proteins of approximately 74 kD (p74) and 78 kD (p78) that coimmunoprecipitated with tropoelastin. Microsequencing of peptide fragments from a cyanogen bromide digest of p78 identified this protein as BiP and sequence analysis identified p74 as the peptidyl-prolyl cis-trans isomerase, FKPB65. The appearance of BiP and FKBP65 in the immunoprecipitations could be enhanced by the addition of brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) to the culture medium for the final 4 h of labeling. Tropoelastin accumulates in the fused ER/Golgi compartment in the presence of BFA if its degradation is inhibited by ALLN (Davis, E.C., and R.P. Mecham. 1996. J. Biol. Chem. 271:3787-3794). The use of BFA and other secretion-disrupting agents suggests that the association of tropoelastin with FKBP65 occurs in the ER. Results from this study provide the first identification of a ligand for an FKBP in the secretory pathway and suggest that the prolyl cis-trans isomerase activity of FKBP65 may be important for the proper folding of the proline-rich tropoelastin molecule before secretion.

Amino Acid Isomerases↗

PubMed1995

Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development.

The production of elastin, an essential extracellular matrix protein of terminal airway interstitium, occurs mostly during early development. Because glucocorticoids influence airway maturation, we studied the effect of dexamethasone (Dex) on tropoelastin expression during fetal lung development. Timed-pregnant rats were treated with Dex (1 mg/kg daily), and fetal lungs were collected 3 days later at 17, 19, and 21 days of gestation. Dex treatment resulted in about a threefold increase in tropoelastin mRNA levels at 19 days concomitant with accelerated airway development. By in situ hybridization, Dex treatment increased the number of tropoelastin-expressing cells and the level of tropoelastin mRNA per cell. In organ culture, Dex increased lung tropoelastin expression and augmented cortisol stimulation of tropoelastin expression. In fetal pulmonary artery smooth muscle cells, 10(-8) M Dex upregulated tropoelastin mRNA expression and increased tropoelastin promoter-chloramphenicol acetyl transferase activity in transient transfections. These data indicate that pharmacologically administered glucocorticoids transcriptionally upregulate fetal lung tropoelastin expression and suggest that steroid hormones may be important regulators of elastin production in vivo.

Animals↗

PubMed1991

Hypoxia-induced inhibition of tropoelastin synthesis by neonatal calf pulmonary artery smooth muscle cells.

Animals chronically exposed to hypoxia develop characteristic structural changes in the pulmonary arterial vasculature including cell hypertrophy, hyperplasia, and increased deposition of extracellular matrix proteins. The medial smooth muscle cells' (SMC) increase in tropoelastin mRNA expression and elastin deposition as determined by in situ hybridization and histologic examination appears to contribute significantly to this increase in matrix protein accumulation. The primary stimulus for the increased tropoelastin production, which persists in vitro, is unknown but mechanical forces and hypoxia seem to play a role. In order to determine the direct effects of hypoxia on tropoelastin production by pulmonary artery SMC, cultured neonatal bovine pulmonary artery SMC were exposed to 3%, 10%, and 21% O2 concentrations for 48, 72, and 120 h and soluble tropoelastin was measured by direct immunoassay. Tropoelastin mRNA levels were also determined by Northern and slot blot analysis after 48 h of incubation under hypoxic conditions. SMC cultured in 3% and 10% O2 for 120 h showed dose-dependent decreases (11-fold and 2-fold, respectively) in measured tropoelastin levels compared with SMC cultured in 21% O2 conditions. This decrease was not due to cell damage or accumulation of toxic metabolites while under hypoxic conditions nor to a change in tropoelastin partitioning between the cell and media. Tropoelastin mRNA levels were also decreased under hypoxic conditions. Secreted, cell layer, and total protein synthesis determined by L-[3H]leucine incorporation again showed a dose-dependent decrease under hypoxic conditions but not to the same extent as tropoelastin production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Chick tropoelastin isoforms. From the gene to the extracellular matrix.

Studies from several laboratories have demonstrated the existence of multiple tropoelasting mRNAs and protein isoforms. The present study was designed to examine the developmental expression of a specific tropoelastin mRNA, its encoded isoform, and the fate of that isoform in the extracellular matrix. A chick genomic DNA library was screened with a chick tropoelastin cDNA. Seven unique, overlapping clones spanning 39 kilobases were isolated. A synthetic oligonucleotide complementary to a variable tropoelastin mRNA sequence was used to identify a 1.5-kilobase PstI-BamHI genomic fragment. Nucleotide sequence data revealed that the putative exon was surrounded by intron sequences possessing canonical splice sites at the exon/intron borders. Using both immunologic and molecular probes specific to the tropoelastin isoform and mRNA, quantitative protein and RNA analyses were performed. Results demonstrate that total tropoelastin mRNAs increased significantly during aortic embryogenesis whereas the amount of mRNA containing the variable exon remained relatively constant. The amount of total tropoelastins within the same developmental period reflect the level of total tropoelastin mRNA. The amount of the tropoelastin isoform containing the variable exon essentially mirrored the corresponding mRNA with the exception that a decrease in the isoform at day 15 was not seen in the mRNA level. Immunoelectron micrographs of 13-day chick aortic tissue using both total and isoform-specific antisera showed ultrastructural localization to definable elastic fibers. Antibodies to the variable tropoelastin isoform occurred preferentially at sites where elastic fiber microfibril structures were evident.

Amino Acid Sequence↗

PubMed1998

Generation of a tropoelastin mRNA variant by alternative polyadenylation site selection in sun-damaged human skin and ultraviolet B-irradiated fibroblasts.

The goal of this research was to delineate the post-transcriptional mechanisms responsible for the increased elastin synthesis characteristic of sundamaged skin. In this study, a unique molecular variant of the tropoelastin mRNA transcript was identified in human sundamaged skin that was derived from the usage of an alternate polyadenylation site. Nonsolar exposed human skin expressed one tropoelastin mRNA species whereas sundamaged human skin expressed the primary tropoelastin mRNA and a larger, alternate tropoelastin mRNA formed from the utilization of a second polyadenylation site. Cultured human skin fibroblasts expressed both tropoelastin transcripts and in vitro UV treatment increased the amount of the unique tropoelastin mRNA. Hairless mouse skin (normal and UV treated) expressed the primary tropoelastin transcript although UV irradiation increased the length of its poly (A) tail two-fold. Therefore, UV radiation may stimulate elastin production by affecting polyadenylation site selection and the poly (A) tail length of tropoelastin mRNA.

Aged↗

PubMed1980

Comparison of aortic and ear cartilage tropoelastins isolated from lathyritic pigs.

Tropoelastin was isolated from aortae and auricular cartilage obtained from lathyritic piglets. The two tissue-specific tropoelastins were judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on a high-pressure liquid chromatograph. Comparative studies of the tropoelastins were performed. Amino acid analysis revealed that the aortic and cartilage tropoelastins were very similar, if not identical, with the only exception that the cartilage tropoelastin contained more hydroxyproline and less lysine residues, both of which can be attributable to post-translational modifications. Both tropoelastins possess an apparent molecular weight of 70 000 and exhibit similar peptide fragments with limited trypsin cleavage. Antiserum raised to the aortic tropoelastin was used to show immunological identity between the two tissue tropoelastins.

Amino Acid Sequence↗

PubMed1995

Enhanced tropoelastin-degrading activity during cell passages in cultured smooth muscle cells.

Tropoelastin expression in vascular smooth muscle cells during serial cell passages from the primary to the tertiary culture was studied. The level of tropoelastin was found to be greatly reduced as the number of cell passages increased. The translational activity and level of elastin mRNA were essentially unchanged throughout the cell passages. The reduction in tropoelastin expression was not due to the repetitive trypsin treatment nor to the prolyl hydroxylation level of the newly-synthesized elastin. A comparable decline in tropoelastin expression was also found with increasing cell division in the primary cultures plated at different cell densities. A pulse-chase experiment revealed that the newly-synthesized elastin in the tertiary culture degraded more rapidly than that in the primary culture. The culture medium harvested from the tertiary culture exhibited a higher tropoelastin-degrading activity than that from the primary culture in the test-tube. The degrading activity of the tertiary culture was inhibited by the addition of 1 mM ethylenediaminetetraacetic acid or 1 mM phenylmethylsulfonyl fluoride, but not by 1 mM N-ethylmaleimide. These results suggest that the reduction in tropoelastin expression during the cell passages from the primary to the tertiary culture is due to the enhanced tropoelastin-degrading activity of the tertiary culture. The transition to tropoelastin-degrading phenotype during cell passages may explain the biological mechanisms of smooth muscle cell migration from the media to the intima observed in the pathological conditions.

Animals↗