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

Abstract

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...

Non Insulin Dependent Diabetes in Sand Rat (Psammomys obesus) and Production of Collagen in Cultured Aortic Smooth Muscle Cells. Influence of Insulin

“…[29,30,31] In fact, the fibrosis of the aortic wall would be due to the presence of collagen, [32] which is dominant in the proliferative intima. [33] Our qualitative and quantitative study on the phenotypic expression of collagen has shown that cultured SMCs of the normal and diabetic Psammomys obesus synthesise more type I than type III collagen, in accord with the results of Layman et al [34] [36] have shown that SMC produced essentially collagen I and III. Ang et al [37] have observed an activation of mRNA coding 1 (I) and czl (III) chains of procollagen in rabbit synthetic SMCs. Compared with control, the diabetic state induced a large increase in type I and type III collagen biosynthesis and secretion.…”

Overloading human aortic smooth muscle cells with low density lipoprotein-cholesteryl esters reproduces features of atherosclerosis in vitro.

“…The electron microscopic appearance of the cultured human aortic smooth muscle cells used in the current experiments was similar to that reported previously for human (23) and monkey aortic smooth muscle cells (24). In particular, the human smooth muscle cells showed abundant myofilaments (Fig.…”

Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens.

“…They do not contract in response to angiotensin II or norepinephrine or to electrical or mechanical stimulation (7,17,18) but readily synthesize collagen, elastin, and glycosaminoglycans (1,2,16,22,23,32) and are thus in a synthetic state . Subcultured aortic smooth muscle cells exposed to the platelet-derived growth factor (PDGF) in whole blood serum (WBS) proliferate in a dose-dependent manner with cell division commencing within 24 to 36 h of challenge (25)(26)(27)(28), and smooth muscle which has migrated from aortic explants, proliferated, and reached a stationary growth phase can be stimulated to another proliferative phase by increased amounts of PDGF, hyperlipemic serum, or its low-density lipoprotein (LDL) within 1-2 d of challenge (8)(9)(10)(11)) .…”