The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells.

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.     

Morphological and functional differentiation of cultured vascular smooth-muscle cells

Summary In numerous investigations using cultured smooth-muscle cells, investigators have consistently added 10–20% fetal calf serum (FCS) to the medium to maintain viable cells. In the present study we utilized an optical technique to investigate whether smooth-muscle cells, cultured with or without FCS, maintain their contractile activity in vitro. With such optical measurement, we were able to detect signals due to spontaneous contractions, in muscle cells cultured in FCS-free medium for up to 8 days, and, for the first time, were also able to observe the conduction of these cell contractions.The ultrastructural characteristics of cultured smooth-muscle cells during contractile activity, were also examined by electron microscopy. The cells were mature and well-differentiated, and were packed with numerous myofilaments. They had developed long cell processes, and were linked to one another by gap junctions.These observations indicated that the smooth-muscle cells, cultured without FCS for 7 to 8 days, were morphologically mature and maintained their contractile activity, whereas the cells cultured in FCS-containing medium showed no detectable signs of contractile activity.     

Regulation of protein accumulation in cultured cells.

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.     

Isolation of alpha-amino adipic acid from mature dermal collagen and elastin. Evidence for an oxidative pathway in the maturation of collagen and elastin.

Human serum alpha-2-macroglobulin has been found to be a major cadmium-binding protein $\text{in vitro}$. Serum and alpha-2-macroglobulin equilibrated with cadmium at the 0.20 ppm level were chromatographed over Sephadex and agarose gels to separate and estimate the molecular weights of the proteins. Alpha-2-macroglobulin was found to fragment into reproducible fragments when chromatographed on agarose gels showing different metal-binding fractions for cadmium and endogenous zinc. The distribution of cadmium on serum protein chromatograms was correlated with alpha-2-macroglobulin chromatograms. Cadmium was bound to fractions with molecular weights as high as 800,000 daltons with an affinity greater than that observed for serum albumin.     

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells

Previously we have shown that hyperosmolarity increasesNa⁺-myo-inositolcotransporter (SMIT) activity and mRNA levels in cultured endothelialcells. Because hyperosmolarity and cytokines, such as tumor necrosisfactor- (TNF-), activate similar signal transduction pathways, weexamined the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation. In contrastto the effect of hyperosmolarity, TNF- caused a time- andconcentration-dependent decrease in SMIT mRNA levels andmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was found in large-vessel endothelial cells (derived fromthe aorta and pulmonary artery) and cerebral microvessel endothelialcells. In bovine aorta and bovine pulmonary artery endothelial cells,TNF- activated nuclear factor (NF)-B. TNF- also increasedceramide levels, and C₂-ceramidemimicked the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation in bovineaorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibitNF-B activation, partially prevented the TNF--induced decrease inmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was also partially prevented by the protein kinase Cinhibitor calphostin C but not by staurosporine. These studiesdemonstrate that TNF- causes a decrease in SMIT mRNA levels andmyo-inositol accumulation in culturedendothelial cells, which may be related to the activation of NF-B.

     

Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition

Diabetic nephropathy, one of the microvascular complications of diabetes mellitus, is a leading cause of end-stage renal disease. Berberine is one of the main constituents of Coptidis Rhizoma and Cortex Phellodendri. In this study, we investigated the effects of berberine on fibronectin and collagen production, and explored the role of p38MAPK signaling pathway in rat glomerular mesangial cells cultured under high glucose condition. Six groups were divided according to the different experimental conditions: (1) Normal glucose group (NG); (2) Mannitol group (Mannitol); (3) High glucose group (HG); (4) SB203580 treatment group (HG + SB203580); (5) Berberine low dosage group (HG + BBR 30 μM); (6) Berberine high dosage group (HG + BBR 90 μM). Cell proliferation and collagen synthesis were measured by MTT and ³H-proline incorporation assay, respectively. The phospho-p38MAPK, phospho-cAMP response element binding protein (CREB) and fibronectin were detected by western blot analysis. Fibronectin protein expression and collagen synthesis were significantly increased in HG-treated group compared with normal glucose group (P < 0.05). In SB203580 treatment group and two groups of berberine, protein expression of fibronectin and collagen synthesis were obviously decreased compared with HG-treated group (P < 0.05). Berberine significantly decreased protein expression of fibronectin compared with SB203580 treatment group (P < 0.05). Berberine at high dosage significantly decreased collagen synthesis compared with SB203580 treatment group (P < 0.05). Both SB203580 and berberine significantly decreased phospho-p38MAPK and phospho-CREB level compared with HG-treated group (P < 0.05). These results indicated that berberine might inhibit fibronectin and collagen synthesis partly via p38MAPK signal pathway in rat glomerular mesangial cells exposed to high glucose.     

Characterization of ectonucleotidases on vascular smooth-muscle cells.

We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5'-[beta-thio]triphosphate, but not of adenosine 5'-[alpha-thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5'-[alpha-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5'-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5'-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5'-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present.     

Ehrlich chromogens, probable cross-links in elastin and collagen.

(1) Proteolytic digests of tissue elastin contain material which reacts with dimethylaminobenzaldehyde in acid solution (Ehrlich's reagent) to give a cherry-pink colour. This Ehrlich chromogen(s) [EC(s)] is similar to but not identical with EC(s) previously demonstrated in tissue collagens [Scott, Hughes & Shuttleworth (1979) Biosci. Rep. 1, 611-618]. Both ECs react with diazonium salts in acid to give coloured products. (2) Diazobenzene linked via a phenolic ester to polyacrylamide beads (Biogel P10) has been used to absorb ECs specifically and almost quantitatively from proteolytic digests. The coupled deeply coloured azo-EC-peptides were then recovered after mild alkaline cleavage from the support and purified by gel chromatography. (3) Using 15N-labelled NaNO2, the collagen azo-EC-peptides were prepared, and 15N abundance measured therein. The molar absorption coefficient of the azo-EC group was calculated (18,700) based on the assumption that each azo-EC group contained one 15N atom. (4) Collagen azo-EC-peptides contained glucose and galactose, whereas elastin azo-EC peptides did not. The amino acid patterns of the two peptides were quite different, the former being rich in polar amino acids, the latter containing much alanine. The patterns were compatible with an origin from the cross-linking regions of collagen and elastin respectively. (5) Quantitative (molar) comparisons of the azo-EC group content with amino acid, amino end-group and sugar contents, and azo-EC peptide molecular mass, suggest that a structure is present in the collagen azo-EC-peptides containing two EC groups shared between four peptide chains. Three peptide chains probably meet at each (cross-linking) EC group. (6) Based on this structure, about 15% of adult bovine skin collagen contains EC groups.     

Processing of soluble elastin in cultured neonatal rat smooth muscle cells

The synthesis and extracellular deposition of elastin by cultured neonatal rat aorta smooth muscle cells has been followed. The addition of beta-aminopropionitrile to the culture medium promotes accumulation of soluble precursors of elastin. Under such conditions, a protein possessing characteristics of a soluble elastin precursor with an apparent molecular weight of 77,000 was detected and partially purified. Pulse-chase studies suggested that this 77-kDa protein undergoes an extracellular, enzymatically catalyzed process to a 71-kDa protein. This 71-kDa protein is strikingly similar to tropoelastins isolated from other tissue systems, in which no evidence for higher molecular weight soluble precursors is at present available. Data presented in this communication suggest that the 77-kDa protein, which we have designated protropoelastin, represents a precursor to the tropoelastin moiety produced in the neonatal rat smooth muscle cell culture.     

The effect of donor age on the in vitro life span of cultured human arterial smooth-muscle cells

Summary The number of population doublings of cultured human arterial smooth-muscle cells decreased as a function of donor age (0.5 to 82 years). Cells from older donors als showed longer latent periods for outgrowth from explants. These results extend other comprable observations with human skin fibroblasts to another cell type, and may have relevance to the pathogenesis of atherosclerosis with aging in vivo. This study was supported by a reserach program project grant (AG00299) from the National Institutes of Health.     

Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth-muscle cells

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell-free systems and ANP-stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor-coupled guanylate cyclase activation, other second-messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium 125I-ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low-affinity (approximately 10(-8) M, 84.5 fmol/10(5) cells) and high-affinity (approximately 10(-10) M, 12.5 fmol/10(5) cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC50 approximately 10(-8) M) and inhibits agonist-(isoproterenol and forskolin)-induced increases in intracellular cyclic AMP (IC50 approximately 10(-9) M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half-maximally effective concentration of approximately 10(-10) M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd-Ins) (200%). Ptd-Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [3H]arachidonic acid label in response to ANP (EC50 approximately 10(-10) M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A2 (generation of releasable [3H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.     

Raman scattering of collagen, gelatin, and elastin.

The Raman spectra of collagen, gelatin, and elastin are presented. The Raman lines in the latter two spectra are assigned by deuterating the amide N-H groups in gelatin and by studying the superposition spectra of the constituent amino acids. Two lines appear at 1271 and 1248 cm^?1 in the spectra of collagen and gelatin that can be assigned to the amide III mode. Possibly, the appearance of two amide III lines is related to the biphasic nature of the tropocollagen molecule, i.e., proline-rich (nonpolar) and proline-poor (polar) regions distributed along the chain. The melting, or collagen-to-gelatin transition, in water-soluble calf skin collagen is studied and the 1248-cm^?1 amide III line is assigned to the 3₁ helical regions of the tropocollagen molecule. Elastin is thought to be mostly random and the Raman spectrum confirms this assertion. Strong amide I and III lines appear at 1668 and 1254 cm^?1, respectively, and only weak scattering is observed at 938 cm^?1. These features have been shown to be characteristic of the disordered conformation in proteins.