Stimulation Of Hyaluronic Acid Synthesis Research Articles

Glucosamine: an ingredient with skin and other benefits

Both glucosamine and its derivative N-acetyl glucosamine are amino-monosaccharides that serve key biochemical functions on their own and as substrate precursors for the biosynthesis of polymers such as glycosaminoglycans (e.g., hyaluronic acid) and for the production of proteoglycans. Glucosamine has an excellent safety profile and has been shown to provide benefits in several clinical disorders. Glucosamine compounds have been reported to have several beneficial effects on the skin or skin cells. Because of its stimulation of hyaluronic acid synthesis, glucosamine has been shown to accelerate wound healing, improve skin hydration, and decrease wrinkles. In addition, as an inhibitor of tyrosinase activation, it inhibits melanin production and is useful in treatment of disorders of hyperpigmentation. Mechanistically, glucosamine also has both anti-inflammatory and chondroprotective effects. Clinical trials have shown benefit in using oral glucosamine supplementation to improve symptoms and slow the progression of osteoarthritis in humans. Glucosamine has also been used to prevent and treat osteoarthritis in animals. Based on other observations, glucosamine has been suggested for additional clinical uses, including treatment of inflammatory bowel disease, migraine headaches, and viral infections. The current clinical uses for topical and oral glucosamine compounds and the mechanistic rationale for these uses are reviewed here.

Cutaneous restructuration by apple seed phytosterols: from DNA chip analysis to morphological alterations

Plant secondary metabolites such as flavonoids, isoflavones and phytosterols have been proposed as cosmetic ingredients displaying anti‐aging effects. On the cellular level, however, the activity profiles of these ingredients are only partially understood. In this study we analyzed the effects of apple seed phytosterols on age‐related structural and functional parameters using cell biochemical, molecular biological and bioengineering techniques. The expression of age‐related genes was studied using skin equivalents and cDNA microarrays. Incubation of skin equivalents with apple seed phytosterols had significant consequences: (i) differential regulation of a set of genes associated with keratinocyte proliferation and differentiation, (ii) stimulation of hyaluronic acid synthesis, and (iii) increase of epidermal thickness. In vivo studies revealed that apple seed phytosterols improve skin elasticity and decrease skin roughness. In conclusion, apple seed phytosterols display distinct biological effects and significantly improve the structure and function of mature skin.

Verminderte Stimulation der Glycosaminoglycansynthese von kultivierten humanen Hautfibroblasten im Rahmen der In-vitro-Alterung durch Interleukin-1

Addition of human recombinant interleukin-1 (IL-1 alpha or IL-1 beta) to culture medium (supplemented MEM without or with 1%, 10%, or 20% fetal calf serum (FCS)) of human skin fibroblasts exerted a stimulating effect in a dose-dependent manner on glycosaminoglycan (GAG) synthesis, including hyaluronic acid synthesis, of young (Phase II) skin fibroblasts in concentrations of 4, 20, or 100 pg/ml. Stimulation was due to increase in total GAGs, namely extracellular (secreted into culture medium) and cell-bound (pericellular) GAGs. Stimulation with 100 or 200 pg/ml IL-1 beta led to a relative increase (total GAGs: 100%) in hyaluronic acid from 49% to 64 (-FCS) and from 79% to 92% (+10% FCS), respectively. The increase in total GAG synthesis was mainly due to increased synthesis of hyaluronic acid. Maximum stimulation, with and without FCS, was reached by 100 pg/ml IL-1 beta. Compared to young (Phase II) cells senescent (Phase III) cells showed significantly diminished stimulation of hyaluronic acid synthesis by IL-1 beta. Both, IL-1 alpha and IL-1 beta, showed no influence on DNA synthesis. These results suggest that both, IL-1 beta and IL-1 alpha, stimulate GAG and especially hyaluronic acid synthesis, but not DNA synthesis, in vitro. The diminished response of GAG and hyaluronic acid synthesis during ageing of these in vitro cultured fibroblasts gives support to the hypothesis that similar processes might occur during ageing in organisms.

Hyaluronic acid synthesis by mural granulosa cells and cumulus cells in vitro is selectively stimulated by a factor produced by oocytes and by transforming growth factor-beta.

In ovarian antral follicles cumulus cells (approximately 1,000/follicle) closely surround the oocyte, and mural granulosa cells (approximately 50,000/follicle) are distributed at the periphery. Previous work (Salustri, A., Yanagishita, M., and Hascall, V. C. (1990) Dev. Biol. 138, 26-32) showed that oocytes produce a factor(s) which stimulates hyaluronic acid (HA) synthesis by cumulus cells during expansion of the cumulus cell-oocyte complex. We now show that mural granulosa cells also respond in vitro to the oocyte factor(s) with greatly increased HA synthesis. As with cumulus cells, a factor(s) present in fetal calf serum is required to retain newly synthesized HA in the extracellular matrix. Unlike cumulus cells, follicle-stimulating hormone (FSH) is not required for maximal stimulation, in part because mural granulosa cells synthesize prostaglandin E2 which can substitute for FSH in promoting cumulus cell-oocyte complex expansion. Of several growth factors studied, only transforming growth factor-beta 1 (TGF-beta 1) stimulated HA synthesis in both cell types. However, the stimulation of HA synthesis by TGF-beta 1 was additive with that for the oocyte factor(s), and neutralizing antibodies to TGF-beta did not inhibit the response to the oocyte factor(s). The results indicate that the oocyte factor(s) and TGF-beta 1 are not the same and that they operate through different receptors in stimulating HA synthesis. Epidermal growth factor was able to replace FSH in amplifying the response of cumulus cells to the oocyte factor(s) and in stimulating synthesis of dermatan sulfate proteoglycans.

Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: Involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146–153]. In the present study, the interaction of [ 3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [ 3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [ 3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P < 0.05- P < 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca 2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.

The effect of interleukin-1 beta on hyaluronic acid synthesized by adult human gingival fibroblasts in vitro.

The effect of recombinant interleukin‐1β (IL‐lβ) on hyaluronic acid synthesis by human gingival fibroblasts was studied. IL‐1bT caused a dose‐dependent increase in the incorporation of (3lucosamine into hyaluronic acid. The 35S/35H ratios of labeled macromolecules did not change regardless of the presence or absence of TL‐lβ and indicates stimulation of hyaluronic acid synthesis. Inhibition of cell proliferation by hydroxyurea caused an increase in hyaluronic acid synthesis. The effect of IL‐1β on hyaluronic acid synthesis in the presence of hydroxyurea was increased over untreated and IL‐lβ‐treated controls, but equivalent to the hydroxyurea‐treated controls. Thus the effect of IL‐1β on hyaluronic acid synthesis may be independent of cell proliferation. Furthermore, inhibition of prostaglandin E2 synthesis by indomethacin abolished the effect of IL‐1β on hyaluronic acid synthesis. Inhibition of new protein synthesis by cycloheximide negated the effect of IL‐β on hyaluronic acid synthesis. This may be related to inhibition of new hyaluronate synthetase synthesis, since IL‐1β stimulated the level of hyaluronate synthetase activity. Sepharose CL‐2B chromatography revealed that most of the newly synthesized hyaluronic acid was of large molecular size. The cells exposed to IL‐1β retained more large molecular size hyaluronic acid in their cell layer environment than did the control cells. These responses by fibroblasts to IL‐1β may be indicative of early tissue repair.

The synthesis of hyaluronic acid by human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment.

Various cell lines of human synovial fibroblasts derived from synovium obtained at the time of biopsy or total joint-replacement surgery have been established. The synthesis of 3H-labelled hyaluronic acid (HA) in these cells has been determined, and the effects of adding HA of varying molecular size to the cultured cells examined. The results obtained clearly show that the in vitro synthesis of HA by these cells is influenced by the concentration and molecular weight (MW) of the HA in their extracellular environment. Synovial fibroblasts derived from an osteoarthritic joint demonstrated the most marked response on exposure to exogenous HA, showing a stimulation of HA synthesis with preparations of weight-average molecular weight (Mw) greater than 5 X 10(5) in a concentration dependent manner. HA preparations with Mw less than 5 X 10(5) showed little or no effect except at high concentrations where a suppression of biosynthesis was observed. A model to explain these findings is proposed.

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [ 14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [ 14C]glucosamine. The major part of [ 14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.

Metabolic change in cultured gingival fibroblasts exposed to bacterial extracts. Stimulation of hyaluronic acid synthesis.

Hyaluronic acid synthesis was studied in human gingival fibroblast cultures exposed to bacterial extracts from dental plaque, Bacillus cereus, and Escherichia coli (lipopolysaccharide, LPS). Dental plaque extract was the best stimulant of hyaluronic acid synthesis. B. cereus extract was almost as effective, but the effect of LPS was insubstantial. This indicates that gram‐positive microorganisms of dental plaque, as well as gram‐negative bacteria, may be involved in the stimulation of hyaluronic acid synthesis in gingival fibroblasts.The effect of indomethacin, cortisol, and cycloheximide on hyaluronic acid synthesis were studied in control and plaque extract treated cultures. Indomethacin had no effect whereas cortisol inhibited the synthesis both in control and treated cultures. Cycloheximide prevented the stimulation of hyaluronic acid synthesis in treated cultures. This suggests that protein synthesis is required for the stimulation of hyaluronic acid synthesis in the presence of plaque extract. The altered metabolism of connective tissue cells due to dental plaque may be important in the development of periodontal disease.

The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts.

Treatment of chicken embryo fibroblasts (CEF) with the tumor promoter, phorbol myristate acetate (PMA), resulted in a rapid increase in their ability to synthesize the glycosaminoglycan, hyaluronic acid (HA), whereas the parent compound, phorbol, had no effect. CEF cultures incubated with PMA (100 ng/ml) for 6 h resulted in a 15-fold increase in HA synthetase activity compared with phorbol-treated control cultures. The incorporation of [3H]acetate into HA and chemical determination of this polymer also demonstrated increased synthesis of HA by cells treated with PMA. We have previously shown that CEF infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24, exhibit increased synthesis of HA upon transformation. PMA treatment of cells transformed with RSV-LA24 results in a further 1.5-fold stimulation of HA synthesis. These data indicate that PMA causes an increased synthesis of HA in CEF which is analogous to the increased synthesis of HA found in virally transformed CEF.

Stimulation of palatal glycosaminoglycan synthesis by cyclic AMP

Intracellular levels of cyclic AMP in primary cultures of mouse embryo palate mesenchyme cells were elevated by exogenous administration of dibutyryl cAMP, 8Br-cAMP, prostaglandin E 2 or prostacyclin. Glycosaminoglycan synthesis was stimulated in a dose-dependent manner. Qualitative analysis by DEAE anion exchange chromatography and sensitivity to hyaluronidase digestion indicated preferential stimulation of hyaluronic acid synthesis. Cyclic AMP may thus play a role in regulating the synthesis of palatal glycosaminoglycans known to be requisite for normal development of the palate.

Induction of hyaluronic acid synthetase by estrogen in the mouse skin

Hyaluronic acid synthetase activity was measured in male mouse skin following the topical application of estradiol in vivo. The enzyme activity increased in parallel with the hyaluronic acid content of the skin, and showed a similar response in the skin of ovariectomized female mice. The increase in enzyme activity was reduced by the anti-estrogen agents, tamoxifene citrate and clomiphene citrate, which block competitively the binding of estrogen to the estrogen receptor. The increase in hyaluronic acid synthetase activity was also reduced by topical application of cycloheximide or by subcutaneous injection of actinomycin D. The results suggest that the stimulation of hyaluronic acid synthesis in mouse skin in response to estrogen treatment is mediated through estrogen receptors and involves the induction of the enzyme hyaluronic acid synthetase.

Mechanisms by which Retrobulbar Fibroblasts are Stimulated by Lymphocytes: Role of Cyclic Nucleotide

SummaryIt was shown that lymphocytes and lymphocyte products stimulated hyalu-ronic acid (HA) synthesis in cultures of human retrobulbar fibroblasts. This stimulation was simulated by dibutyryl cyclic AMP (DbcAMP), 2 × 10-4-10-2M, and potentiated by theophylline, 10-4M. Lymphocytes induced a significant increase in the cAMP concentration within the fibroblasts. These data support the concept that the lymphocyte stimulation of HA synthesis is mediated by cAMP.However, lymphocytes induced greater glucose utilization by the cultures than DbcAMP. Also, the intrinsic viscosity of the HA was decreased by lymphocytes but increased by DbcAMP. Lymphocytes probably produce more effects in the fibroblasts than those mediated by cAMP.Hydrocortisone inhibited both the cAMP and the HA responses to lymphocytes but potentiated the stimulation of HA synthesis by DbcAMP. Hydrocortisone thus appears to interfere with the earliest effects of lymphocytes, possibly inhibiting the binding of the active factor in lymphocytes to ...