Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment

doi:10.1016/0304-4165(85)90125-4

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 840, Issue 2, 18 June 1985, Pages 245-254

https://doi.org/10.1016/0304-4165(85)90125-4 Get rights and content

Abstract

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately $13$ of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

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Interestingly, l-ascorbic acid (AA), a potent stimulator of collagen production [24–26], has also been listed as an inhibitor of elastin deposition. It has been suggested that AA may destabilize tropoelastin mRNA [27–29] and cause overwhelmed hydroxylation on prolyl/lysyl residues of tropoelastin molecules, thereby promoting their intracellular accumulation and inhibiting their secretion [30]. Our present studies explored the elastogenic potential of the l-ascorbic acid derivative (+) sodium l-ascorbate (SA) in experimental models utilizing primary cultures of skin fibroblasts, fat-derived fibroblasts and cultures of dermal explants derived from normal and pathologic human skin.
Vitamin C (l-ascorbic acid), a known enhancer of collagen deposition, has also been identified as an inhibitor of elastogenesis. Objective Present studies explored whether and how the l-ascorbic acid derivative (+) sodium l-ascorbate (SA) would affect production of collagen and elastic fibers in cultures of fibroblasts derived from normal human skin and dermal fat, as well as in explants of normal human skin, stretch-marked skin and keloids.Methods Effects of SA on the extracellular matrix production were assessed quantitatively by PCR analyses, western blots, biochemical assay of insoluble elastin and by immuno-histochemistry. We also evaluated effects of SA on production of the reactive oxygen species (ROS) and phosphorylation of IGF-I and insulin receptors. Results SA, applied in 50–200 μM concentrations, stimulates production of both collagen and elastic fibers in all tested cultures. Moreover, combination of SA with a proline hydroxylase inhibitor induces a beneficial remodelling in explants of dermal scars, resulting in the inhibition of collagen deposition and induction of new elastogenesis. Importantly, we revealed that SA stimulates elastogenesis only after intracellular influx of non-oxidized ascorbate anions (facilitated by the sodium-dependent ascorbate transporter), that causes reduction of intracellular ROS, activation of c-Src tyrosine kinase and the enhancement of IGF-1-induced phosphorylation of the IGF-1 receptor that ultimately triggers elastogenic signalling pathway. Conclusion Our results endorse the use of this potent stimulator of collagen and elastin production in the treatment of wrinkled and stretch-marked skin. They also encourage inclusion of SA into therapeutic combinations with collagenogenesis inhibitors to prevent formation of dermal scars and keloids.
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Elastin is also subjected to ascorbate-dependent hydroxylation yet the function of this modification is less well understood. In particular, a number of studies have indicated that ascorbate leads to enhanced collagen production and reduced elastin synthesis (Bergethon et al., 1989; Dunn and Franzblau, 1982; Leesa et al., 1985). However, these studies were limited to analysis of cells grown under traditional 2-dimensional tissue culture conditions.
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Our goal is to fabricate continuous sheets of elastin atop non-biodegradable hydrogels (hylans) containing crosslinked hyaluronan, a glycosaminoglycan. Such elastin–hyaluronan composites may be useful to tissue engineer replacements for the glycosaminoglycan- and elastin-rich layers of the native aortic valve cusp. Neonatal rat aortic smooth muscle cells were cultured atop hylan gels with micro-textured surfaces, and on plastic, and the components of the extracellular matrix (collagen, elastin) were periodically analyzed.
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Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment

Abstract

Arch. Biochem. Biophys.

Biochim. Biophys. Res. Commun.

Int. Rev. Connect. Tissue Rev.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

In Vitro

Biochemistry