The synthesis of connective tissue protein in smooth muscle cells

Faris, Barbara; Salcedo, Lily L.; Cook, Victoria R.; Johnson, Linda S.; Foster, Jeffrey S.; Franzblau, Carl

doi:10.1016/0005-2787(76)90330-0

Cited by 96 publications

(29 citation statements)

References 12 publications

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“…Preliminary studies by one of us indicated that cultured aortic smooth muscle cells derived from fetal aortas grown in the presence of 10% fetal calf serum synthesized only type I collagen (7). Recently, Faris et al (8) have reported the synthesis of radioactive peptide-bound hydroxyproline by cultured rabbit medial smooth muscle cells. We report here that cultured smooth muscle cells derived from adult human aortas synthesize both type I and type III collagens when grown in 10% adult human serum.…”

mentioning

confidence: 99%

Biosynthesis of type I and III collagens by cultured smooth muscle cells from human aorta.

Layman¹,

Epstein²,

Dodson³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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The synthesis of collagen by human aortic smooth muscle cells was studied after incubating the cells with [3HIproline and [3Hlglycine for 48 hr. The culture medium and cells were lyophilized dthen digested with cyanogen bromide (CNBr) in 70% (wt/vol) formic acid. The resultant peptides were subjected to ion exchange chromatography on CM-cellulose and gel filtration on agarose. On the basis of the molar ratios of the al(Il)CB8 and al(III)CB8 peptides of the al(I) and al (III) chains, approximately one quarter of the collagen synthesized by these cells was identified as type III and three quarters as type I. These data indicate that the smooth muscle can synthesize at least two types of collagen found in the arterial wall. MATERIAL AND METHODS Culture Techniques. Cultures of smooth muscle cells were established from explants of medial smooth muscle obtained from adult human aortas 4-6 hr postmortem. The method for isolating these cells in culture was similar to that of Ross (9), with the exception that the growth medium was supplemented with pooled adult human serum. McCoy's 5a modified medium (Gibco) containing 20% human serum (heat inactivated), 200 units/ml of penicillin, 200 ,g/ml of streptomycin, and 30 units/ml of mycostatin was used for culturing the explanted aortic smooth muscle. Thereafter, 10% human serum was employed for subculturing the smooth muscle cells. Culture medium was changed twice weekly. Only cultures between the third and sixth passage (1:2 split) and exhibiting morphologic 671 and growth characteristics reported for smooth muscle cells were used in these studies.Studies After 48 hr, the medium was removed and lyophilized. The cell layer was scraped from the flask in 20 ml of distilled water and lyophilized. In some experiments both the medium and the cell layer were pooled and lyophilized. The lyophilized samples were digested with CNBr in 70% (wt/vol) formic acid, desalted, and lyophilized as described previously (10). The lyophilized peptides and carrier peptides derived from CNBr digestion of insoluble human placental membranes were redissolved in starting buffer (0.02 M sodium citrate, 0.02 M NaCl adjusted to pH 3.8 with citric acid) and chromatographed on CM-cellulose as previously described (10). Further separation and determination of the relative quantities of CNBr-liberated peptides derived from CM-cellulose chromatography were achieved on a column (1.8 X 230 cm) of agarose (Bio-Gel A-1.5m, 200400 mesh) at room temperature (10). The relative quantities of peptides present were compared by tracing the appropriate peaks onto paper, cutting out the traced outlines, and weighing the individual pieces (10). Some samples were hydrolyzed in 6 M HCI for 24 hr at 1100 and analyzed for radioactive proline and hydroxyproline on a Beckman model 119 amino acid analyzer equipped with a stream-splitting device.Electron Microscopy. For fine structural studies cultured smooth muscle cells were fixed in situ with 3% (vol/vol) glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The cells w...

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mentioning

confidence: 99%

Biosynthesis of type I and III collagens by cultured smooth muscle cells from human aorta.

Layman¹,

Epstein²,

Dodson³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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“…The analysis of the DTT-treated cells is essentially identical with that of the controls. These analyses compare favourably with those for insoluble elastin obtained from intact rabbit aorta (Faris et al, 1976).…”

Section: Resultsmentioning

confidence: 76%

“…Barone et al (1985) Neonatal-rabbit smooth-muscle cells were isolated from aortae of 1-3-day-old New Zealand rabbits as described previously for the neonatal rat (Oakes et al, 1982). Briefly, a collagenase (10 mg; type I, Sigma Chemical Co., St. Louis, MO, U.S.A.)/elastin (2.5 mg; Sigma type III) solution in 20 ml of Dulbecco's modified Eagle's medium without fetal bovine serum (FBS) was added to the medial layer of 20 aortae that were finely minced after careful dissection (Faris et al, 1976). After digestion at 37°C for 45 min the resulting cell suspension was centrifuged at 400 g for 5 min and then washed twice with Dulbecco's medium containing 40 mM-NaHCO3, 200 (v/v) FBS, 100 ,ug of penicillin/ml and 100 ,ug of streptomycin/ml, 0.1 mm non-essential amino acids (Gibco, Grand Island, NY, U.S.A.) and 1 mM-sodium pyruvate.…”

Section: Introductionmentioning

confidence: 99%

Effect of the reducing environment on the accumulation of elastin and collagen in cultured smoothmuscle cells

1989

Biochemical Journal

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We show here that cultured neonatal-rabbit aortic smooth-muscle cells produce and accumulate significant amounts of insoluble elastin. When grown in the presence of ascorbic acid, the amount of insoluble elastin in these cultures decreases, whereas the accumulation of collagen increases. These changes have been attributed to increased hydroxylation of proline in elastin. The function of ascorbic acid in proline hydroxylase, do not demonstrate altered elastin accumulation. These studies are consistent with the molecular iron in the enzyme complex. This study shows that both ascorbic and isoascorbic acids act similarly to modify the accumulation of elastin and collagen in culture. On the other hand, cultures grown in the presence of dithiothreitol, a reducing agent previously shown to act as a cofactor for prolyl hydroxylase, does not demonstrate altered elastin accumulation. These studies are consistent with the suggestion that there is a specific role for ascorbic acid in this cellular system that cannot be replaced by other reducing cofactors. INTRODUCTIONElastin is an insoluble polymeric protein that is responsible for the elastic properties of vertebrate tissues. The insoluble elastin fibre is assembled from soluble precursor molecules termed tropoelastin. Though tropoelastin undergoes few post-translational modifications, one of the best characterized is the hydroxylation of 3-8 %/O of its prolyl residues (Davidson, 1987). The role, if any, of hydroxyproline in tropoelastin is unclear. This is in contrast with hydroxyproline's function in stabilizing the triple-helical confirmation of collagen molecules (Berg & Prockop, 1973;Uitto & Prockop, 1974). L-Ascorbic acid is a cofactor for the enzyme prolyl hydroxylase (EC 1.14.11.2), which catalyses the posttranslational hydroxylation of proline in nascent collagen and elastin molecules (Cardinale & Udenfriend, 1974). In addition, molecular oxygen, ferrous iron and 2-oxoglutarate are necessary cofactors for the normal functioning of proWyl hydroxylase (Adams & Frank, 1980). The biochemical activity of ascorbic acid, a 2-oxolactone, is generally considered to reside in the electroactivity of the keto-enol nature of the C-2 and C-3 atoms. D-Ascorbic acid (isoascorbic acid) is an epimer of Lascorbic acid that differs only by the transposition of the -OH and -H groups at C-5. Since the electroactivity of both molecules is conferred by the keto-enol portion of the ring, both epimers are essentially equivalent in terms of their oxidation-reduction behaviour (Lewin, 1976).The role of ascorbic acid in the mechanism of proline hydroxylation is not completely understood, and is still under investigation (Kiv-irikko & Myllyla, 1987 Udenfriend, 1965;Hutton et al., 1967;Rhoads et al., 1967;Rhoads & Udenfriend, 1970). Later studies with much more highly purified enzyme showed that ascorbic acid was quite specific, though some activity was demonstrated when DTT or L-cysteine was the reductive cofactor (Myllyla et al., 1978). Other investigations have suggested that ascorbic acid...

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“…In these two classes of structural proteins which simultaneously can be synthesized by the same cell population [10,11], each processing event -peptidyl hydroxyproline formation and cross-linkstabilized matrix accumulation -is catalyzed by a specific enzyme, the intracellularly active prolyl 4-hydroxylase [12] and the extracellularly active lysyl oxidase [13], respectively. Both enzymes utilize unmodified elastin and unmodified collagen [12][13][14][15]; experimental evidence for peptide substrate-specific isoenzymes is not available.…”

Section: Introductionmentioning

confidence: 99%

Pyrroloquinoline quinone and molecules mimicking its functional domains Modulators of connective tissue formation?

et al. 1987

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The synthesis of connective tissue protein in smooth muscle cells

Cited by 96 publications

References 12 publications

Biosynthesis of type I and III collagens by cultured smooth muscle cells from human aorta.

Biosynthesis of type I and III collagens by cultured smooth muscle cells from human aorta.

Effect of the reducing environment on the accumulation of elastin and collagen in cultured smoothmuscle cells

Pyrroloquinoline quinone and molecules mimicking its functional domains Modulators of connective tissue formation?

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