James V Gruber1, Robert Holtz2, Jed Riemer3. 1. BotanicalsPlus®, Fairfield, NJ, USA. 2. Bioinnovation® Laboratory, Lakewood, CO, USA. 3. Jeen® International, Fairfield, NJ, USA.
Abstract
BACKGROUND: In the skin, hyaluronic acid is broken down to smaller fragments by hyaluronidase enzymes, particularly when skin is wounded. The impact of various molecular weight fragments of HA on normal human epidermal keratinocytes (NHEK) with regard to expression of important cellular proteins has not been deeply explored. AIMS: Examination of three molecular weight (Mw) fractions of hyaluronic acid: 1) average Mw of the high fraction: 1.5-2 MDa, 2) average Mw of the medium fraction: 200-500 kDa, and 3) average Mw of the low fraction: 5-10 kDa and a unique 1:1:1 composite complex of the three HA fragments (Triluronic® Acid) was done to examine the influence of the HA on two critical skin cell protein targets: hyaluronan synthase-1 (HAS-1) and the HA binding protein cluster of differentiation 44 (CD44). METHODS: NHEKs were treated in vitro with a 1.0% stock solution of each HA Mw fraction at 1.0, 0.5, and 0.1% concentrations of the 1.0% solution and the polysaccharide composite at the same concentrations for 48 Hrs. The cells were than analyzed by ELISA protein assays for HAS-1 and CD44 protein content. RESULTS: Examination of HAS-1 protein expression indicates that none of the HA test materials influenced the expression of HAS-1 at any concentration. Examination of the CD44 protein expression indicated that the low Mw fraction and the commercial complex of the three Mw fractions upregulated CD44 protein expression in NHEKs, but the medium Mw and high Mw HA fractions did not. CONCLUSIONS: In this work, it was demonstrated that HA can influence the expression of CD44 protein, a critical HA transmembrane HA binding protein, and the influence appears to be molecular weight dependent.
BACKGROUND: In the skin, hyaluronic acid is broken down to smaller fragments by hyaluronidase enzymes, particularly when skin is wounded. The impact of various molecular weight fragments of HA on normal human epidermal keratinocytes (NHEK) with regard to expression of important cellular proteins has not been deeply explored. AIMS: Examination of three molecular weight (Mw) fractions of hyaluronic acid: 1) average Mw of the high fraction: 1.5-2 MDa, 2) average Mw of the medium fraction: 200-500 kDa, and 3) average Mw of the low fraction: 5-10 kDa and a unique 1:1:1 composite complex of the three HA fragments (Triluronic® Acid) was done to examine the influence of the HA on two critical skin cell protein targets: hyaluronan synthase-1 (HAS-1) and the HA binding protein cluster of differentiation 44 (CD44). METHODS: NHEKs were treated in vitro with a 1.0% stock solution of each HA Mw fraction at 1.0, 0.5, and 0.1% concentrations of the 1.0% solution and the polysaccharide composite at the same concentrations for 48 Hrs. The cells were than analyzed by ELISA protein assays for HAS-1 and CD44 protein content. RESULTS: Examination of HAS-1 protein expression indicates that none of the HA test materials influenced the expression of HAS-1 at any concentration. Examination of the CD44 protein expression indicated that the low Mw fraction and the commercial complex of the three Mw fractions upregulated CD44 protein expression in NHEKs, but the medium Mw and high Mw HA fractions did not. CONCLUSIONS: In this work, it was demonstrated that HA can influence the expression of CD44 protein, a critical HA transmembrane HA binding protein, and the influence appears to be molecular weight dependent.