Hyaluronic Acid Synthesis Research Articles

Regulation of activated genes of fibroblasts and their improvement of skin aging

Objective To investigate the possible mechanisms using different chemicals to improve the skin aging by the effect of estrogen,vitamin E,vitamin C on the transcriptional level of MMP-1 and HAS-2 mRNAs in cultured human skin fibroblast in vitro.Methods The different concentrations of estrogen,vitamin E,vitamin C were put into cultured human skin fibroblasts.After 24 hours,RT-PCR was used to detect the effects of the different chemicals on the transcriptional levels of the HAS-2 and MMP-1 mRNAs in fibroblasts.Results The effects of estrogen and vitamin C on the MMP-1 mRNA had significant difference between the high dose group and blank control group,and between the low dose group and blank control group (P＜0.05) ; the difference between the low dose group and high dose group was not statistically significant (P＞0.05).The effects of vitamin E and vitamin C on Has-2 mRNA also had significant difference between the high dose group and the blank control group,and between the low dose group and blank control group (P＜0.05),but the difference between the low dose group and high dose group was not statistically significant (P＞0.05).Conclusions Estrogen and vitamin C can reduce the transcription of MMP-1 gene,which decreases the degradation of collagen in the skin; vitamin E and vitamin C can increase the transcription of HAS-2 gene,which may possibly increase the synthesis of hyaluronic acid to alleviate the skin aging. Key words: Matrix metalloproteinase-1 (MMP-1); Hyaluronic acid synthetase-2 (HAS-2); Fibroblast; Skin aging

Dietary Supplement Hymecromone and Sorafenib: A Novel Combination for the Control of Renal Cell Carcinoma

Current treatments for metastatic renal cell carcinoma do not extend survival beyond a few months. Sorafenib is a targeted drug approved for metastatic renal cell carcinoma but it has modest efficacy. Hymecromone is a nontoxic dietary supplement with some antitumor activity at high doses of 450 to 3,000 mg per day. Hymecromone inhibits the synthesis of hyaluronic acid, which promotes tumor growth and metastasis. We recently noted that the hyaluronic acid receptors CD44 and RHAMM are potential predictors of metastatic renal cell carcinoma. In the current study we examined the antitumor properties of hymecromone, sorafenib and the combination in renal cell carcinoma models. Using proliferation, clonogenic and apoptosis assays, we examined the effects of hymecromone (0 to 32 μg/ml), sorafenib (0 to 3.2 μg/ml) and hymecromone plus sorafenib in Caki-1, 786-O, ACHN and A498 renal cell carcinoma cells, and HMVEC-L and HUVEC endothelial cells. A Boyden chamber was used for motility and invasion assays. Apoptosis indicators, hyaluronic acid receptors, epidermal growth factor receptor and c-Met were evaluated by immunoblot. The efficacy of hymecromone, sorafenib and hymecromone plus sorafenib was assessed in the sorafenib resistant Caki-1 xenograft model. Hymecromone plus sorafenib synergistically inhibited proliferation (greater than 95%), motility/invasion (65%) and capillary formation (76%) in renal cell carcinoma and/or endothelial cells, and induced apoptosis eightfold (p <0.001). Hymecromone plus sorafenib inhibited hyaluronic acid synthesis and adding hyaluronic acid reversed the cytotoxicity of hymecromone plus sorafenib. Hymecromone plus sorafenib up-regulated pro-apoptotic indicators and down-regulated Mcl-1, CD44, RHAMM, phospho-epidermal growth factor receptor and phospho-cMet. In all assays hymecromone and sorafenib alone were ineffective. Oral administration of hymecromone (50 to 200 mg/kg) plus sorafenib (30 mg/kg) eradicated Caki-1 tumor growth without toxicity. Hymecromone and sorafenib alone were ineffective. To our knowledge this is the first study to show that the combination of sorafenib and the nontoxic dietary supplement hymecromone is highly effective for controlling renal cell carcinoma.

New hyaluronic acid based brush copolymers synthesized by atom transfer radical polymerization

In this work, an efficient method for the synthesis of hyaluronic acid based brush copolymers using atom transfer radical polymerization (ATRP) has been reported. At first, two different hyaluronic acid (HA) based macroinitiators have been prepared and then they have been used for the polymerization via ATRP of hydrophilic or hydrophobic molecules carrying vinyl portions.In particular, by linking 2-bromo-2-methylpropionic acid (BMP) to the primary hydroxyl groups of tetrabutyl ammonium salt of HA (HA–TBA) or to amino groups of the ethylenediamino derivative of HA–TBA (HA–TBA–EDA), two macroinitiators (HA–TBA–BMP and HA–TBA–EDA–BMP) have been obtained. Then they have been used for the ATRP of poly(ethylene glycol) methacrylate (PEGMA), butyl methacrylate (BUTMA) or N-isopropylacrylamide (NIPAM) using a complex of Cu(I) and 2,2′-Bipyridyl (Bpy), as a catalyst.Both macroinitiators and final copolymers, named as HA–BMP–pPEGMA, HA–BMP–pBUTMA, HA–BMP–pNIPAM, HA–EDA–BMP–pPEGMA, HA–EDA–BMP–pBUTMA and HA–EDA–BMP–pNIPAM, have been characterized by spectroscopic analysis and size exclusion chromatography to confirm the success of the polymerization process.

Synthesis and Characterization of Hyaluronic Acid for Biomedical Application

Hyaluronic acid is a linear polysaccharide composed of glucuronic acid and N-acetylglucosamine. HA-based biomaterials secure wide-range applications in the healthcare industry. The material is found in cartilage, skin, umbilical cord, synovial fluid, as well as in the cell wall of bacteria such a Streptococcus zooepidemicus. As a consequence of these wide applications, HA production from microbial fermentation is receiving increased attention for avoiding the risk of cross-species viral infection, to have lower production cost and to favor a more efficient purification. The molecular weight is increased by adding poly-γ-glutamic acid, which inhibits hyaluronidase of medium, and the degree of purity is enhanced by a purification process using the means of dialysis membrane and organic solvent.

Hyaluronic acid: A key molecule in skin aging.

Skin aging is a multifactorial process consisting of two distinct and independent mechanisms: intrinsic and extrinsic aging. Youthful skin retains its turgor, resilience and pliability, among others, due to its high content of water. Daily external injury, in addition to the normal process of aging, causes loss of moisture. The key molecule involved in skin moisture is hyaluronic acid (HA) that has unique capacity in retaining water. There are multiple sites for the control of HA synthesis, deposition, cell and protein association and degradation, reflecting the complexity of HA metabolism. The enzymes that synthesize or catabolize HA and HA receptors responsible for many of the functions of HA are all multigene families with distinct patterns of tissue expression. Understanding the metabolism of HA in the different layers of the skin and the interactions of HA with other skin components will facilitate the ability to modulate skin moisture in a rational manner.

Functional roles of fructose

During the periimplantation period of pregnancy, pig blastocysts undergo morphological changes and differentiation requiring secretion and transport of nutrients (histotroph) into the uterine lumen. Of these nutrients, glucose is converted to fructose, an isomer of glucose, by conceptus trophectoderm. Although glucose is an energy source for proliferation and growth of mammalian cells, the role of fructose in uterine histotroph is unclear although it is the most abundant hexose sugar in fetal blood and fluids of ungulate mammals (e.g., cows, sheep, and pigs). In this study, we used porcine trophectoderm cells to determine that fructose increased cell proliferation, as did glucose. Western blot analyses of porcine trophectoderm cell extracts revealed that fructose increased the abundance of phosphorylated-RPS6K, -EIF4EBP1, and -RPS6 over basal levels within 30 min, and those levels remained elevated to 120 min. Phosphorylation of both RPS6K and EIF4EBP1 proteins in response to fructose was inhibited by inhibitors of both PI3K and MTOR. Further, when we investigated the inhibition of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) by azaserine (an inhibitor of GFPT1) and GFPT1 siRNA, we found that MTOR-RPS6K and MTOR-EIF4EBP1 signaling in response to fructose is mediated via GFPT1 activation and the hexosamine pathway. We further demonstrated that fructose stimulates the production of hyaluronic acid via GFPT1 and the hexosamine biosynthesis pathway. Collectively, these results demonstrate critical roles for fructose that are mediated via the hexosamine biosynthesis pathway to stimulate MTOR cell signaling, proliferation of porcine trophectoderm cells, and synthesis of hyaluronic acid, a significant glycosaminoglycan in the pregnant uterus.

Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes

Hyaluronic acid (HA), an important component of the extracellular matrix, plays a crucial role for cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor CD44 are expressed in cumulus oocyte complexes (COCs) in different animal species and increase during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. To investigate the effects of the molecular size and concentration of HA on oocyte maturation and further embryo development, bovine oocytes were matured in vitro in the presence or absence of HA, Hyal-2 or 4-methylumbelliferone (4-MU); an HA synthesis inhibitor. The rates of oocyte nuclear maturation to metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Hyal-2 inhibited cumulus cell expansion without affecting oocyte maturation and further embryo development. Whereas, 4-MU at 1 mm reduced cumulus cell expansion, oocyte maturation rate and further embryo development; an effect which was partially abrogated by exogenous HA supplementation. These data suggest that HA production by cumulus cells during maturation is essential not only for cumulus cell expansion, but also for oocyte maturation and further embryo development. This effect is not affected by HA-degradation by Hyal-2.

Abstract 336: 4-methylumbeliferone, an HA synthesis inhibitor, inhibits EMT determinants in bladder cancer cell lines

Abstract INTRODUCTION AND OBJECTIVE: Elevated Hyaluronic Acid (HA) levels are a molecular determinant of bladder cancer (BCa) metastasis. Furthermore, HA, HYAL-1 hyaluronidase and HA-synthesizing enzymes are potential markers for BCa diagnosis and predicting prognosis. 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. Epithelial mesenchymal transition (EMT) is the hallmark of invasion and metastasis, in which cancer cells acquire an invasive phenotype. Since HA promotes tumor metastasis, we evaluated the antitumor effects of 4-MU in vitro and in vivo and examined whether 4-MU can reverse the EMT by inhibiting HA synthesis. METHODS: Quantitative PCR was used to measure mRNA expression of EMT genes (β-catenin, Twist, and Snail) in 66 bladder tissue specimens (27 normal; 39 tumor); follow-up: 26±4.3 months; median 20 months. The effect of 4-MU (0.2 - 0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors and EMT genes were examined in BCa cell lines, 253J-Lung and HT1376. Effect of 4-MU on tumor growth was analyzed in subcutaneous xenografts. RESULTS: Among the EMT genes, both Snail and Twist were differentially expressed in BCa tissues when compared to normal bladder (P&amp;lt;0.001). Furthermore, Twist expression significantly correlated with metastasis (+: 0.25+0.15, -: 0.12+0.14; P=0.028). β-catenin expression negatively correlated with survival (chi square=4.3, p=0.038). 4-MU inhibited proliferation and induced apoptosis in the BCa cells. Following a 48 h exposure at the IC50 for HA synthesis (0.4 mM), 4-MU inhibited proliferation by 61% and increased apoptosis by 73%. 4-MU induced caspase-8, -9 and -3 activation and up-regulation of Fas and FADD. 4-MU downregulated the expression of HA-receptors - CD44, RHAMM, EMT promoters - β-catenin, Snail and Twist by 4-10-fold, but increased the expression of E-cadherin by 2-fold, suggesting EMT-reversal. In xenograft studies, 4-MU significantly decreased tumor growth (&amp;gt; 3-fold) when treatment was started either on the day of tumor cell injection or after tumors became palpable. No weight loss or serum or organ toxicity was observed in treated mice. Tumors showed reduced microvessel density (∼3-fold) and HA expression but increased TURNEL positive cells. CONCLUSION: This is the first study that shows inhibition of HA synthesis by an orally bioavailable, non-toxic supplement potentially reverses EMT and has potent anti-tumor activity. Support: R01 CA 123063-04 (VBL); R01 CA 72821-11 (VBL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 336. doi:1538-7445.AM2012-336

894 4-METHYLUMBELIFERONE A POTENTIAL ANTI-METASTATIC ORAL AGENT FOR BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012894 4-METHYLUMBELIFERONE A POTENTIAL ANTI-METASTATIC ORAL AGENT FOR BLADDER CANCER Michael Garcia-Roig, Travis Yates, Anaid Benitez, and Vinita Lokeshwar Michael Garcia-RoigMichael Garcia-Roig Miami, FL More articles by this author , Travis YatesTravis Yates Miami, FL More articles by this author , Anaid BenitezAnaid Benitez Miami, FL More articles by this author , and Vinita LokeshwarVinita Lokeshwar Miami, FL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.989AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Elevated Hyaluronic Acid (HA) levels are a molecular determinant of bladder cancer (BCa) metastasis. Furthermore, HA, HYAL-1 hyaluronidase and HA-synthesizing enzymes are potential markers for BCa diagnosis and predicting prognosis. 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. Epithelial mesenchymal transition (EMT) is the hallmark of invasion and metastasis, in which cancer cells acquire an invasive phenotype. Since HA promotes tumor metastasis, we evaluated the antitumor effects of 4-MU in vitro and in vivo and examined whether 4-MU can reverse the EMT by inhibiting HA synthesis. METHODS Quantitative PCR was used to measure mRNA expression of EMT genes (β;-catenin, Twist, and Snail) in 66 bladder tissue specimens (27 normal; 39 tumor); follow-up: 26±4.3 months; median 20 months. The effect of 4-MU (0.2 – 0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors and EMT genes were examined in BCa cell lines, 253J-Lung and HT1376. Effect of 4-MU on tumor growth was analyzed in subcutaneous xenografts. RESULTS Among the EMT genes, both Snail and Twist were differentially expressed in BCa tissues when compared to normal bladder (P<0.001). Furthermore, Twist expression significantly correlated with metastasis (+: 0.25+0.15, - : 0.12+0.14; P=0.028). β-catenin expression negatively correlated with survival (chi square=4.3, p=0.038). 4-MU inhibited proliferation and induced apoptosis in the BCa cells. Following a 48 h exposure at the IC50 for HA synthesis (0.4 mM), 4-MU inhibited proliferation by 61% and increased apoptosis by 73%. 4-MU induced caspase-8, -9 and -3 activation and up-regulation of Fas and FADD. 4-MU downregulated the expression of HA-receptors - CD44, RHAMM, EMT promoters - β;-catenin, Snail and Twist by 4-10-fold, but increased the expression of E-cadherin by 2-fold, suggesting EMT-reversal. In xenograft studies, 4-MU significantly decreased tumor growth (> 3-fold) when treatment was started either on the day of tumor cell injection or after tumors became palpable. No weight loss or serum or organ toxicity was observed in treated mice. Tumors showed reduced microvessel density (∼3-fold) and HA expression but increased TURNEL positive cells. CONCLUSIONS This is the first study that shows inhibition of HA synthesis by an orally bioavailable, non-toxic supplement that potentially reverses EMT and has potent anti-tumor activity. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e364 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Garcia-Roig Miami, FL More articles by this author Travis Yates Miami, FL More articles by this author Anaid Benitez Miami, FL More articles by this author Vinita Lokeshwar Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A Stimulatory Thyrotropin Receptor Antibody Enhances Hyaluronic Acid Synthesis in Graves' Orbital Fibroblasts: Inhibition by an IGF-I Receptor Blocking Antibody

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital fat and extraocular muscle tissues and elevated levels of TSH receptor autoantibodies (TRAb). The expansion of orbital tissues involves accumulation of hyaluronic acid (HA) within the orbit. The objective of the study was to determine whether a monoclonal stimulatory TRAb (M22) impacts HA synthesis in GO orbital cells and, if so, whether this might be blocked by an IGF-I receptor (IGF-IR)-blocking antibody (1H7) or inhibitors of various downstream signaling cascades. GO orbital fibroblast cultures (n = 6) were treated with M22, bovine TSH (bTSH), or IGF-I in serum-free medium. Some cultures also received 1H7, LY294002, rapamycin, or protein kinase A inhibitor. HA production and phosphorylated Akt levels in media or immunoblotting for phosphorylated Akt were measured. M22 or bTSH stimulated HA synthesis (2.1-fold with 100 ng/ml M22 and 1.9-fold with 10 U/liter bTSH; P < 0.05 each). M22-induced HA synthesis was inhibited by LY294002 or rapamycin but not by protein kinase inhibitor. HA synthesis stimulated by M22 or IGF-I was inhibited by 1H7 (mean 36.6 ± 5.6% and mean 45.8 ± 7.6%, respectively; P < 0.05 each). Similarly, M22- or IGF-I-stimulated Akt phosphorylation was inhibited by 1H7 (mean 54 ± 9.6 and 36.1 ± 8.8%, respectively; P = 0.01 each). The stimulatory TRAb M22 increases HA production in undifferentiated GO orbital fibroblasts via phosphoinositide 3-kinase/phosphorylated AKT/mammalian target of rapamycin activation. Blockade of IGF-IR inhibits both HA synthesis and Akt phosphorylation induced by M22 or IGF-I in these cells, suggesting that TSH receptor and IGF-IR signaling may be closely linked in the GO orbit.

Pycnogenol® Effects on Skin Elasticity and Hydration Coincide with Increased Gene Expressions of Collagen Type I and Hyaluronic Acid Synthase in Women

Introduction and Objectives: In recent years there has been an increasing interest in the use of nutritional supplements to benefit human skin. Molecular evidence substantiating such effects, however, is scarce. In the present study we investigated whether nutritional supplementation of women with the standardized pine bark extract Pycnogenol® will improve their cosmetic appearance and relate these effects to expression of corresponding molecular markers of their skin. Materials and Methods: For this purpose 20 healthy postmenopausal women were supplemented with Pycnogenol for 12 weeks. Before, during and after supplementation, their skin condition was assessed (i) by employing non-invasive, biophysical methods including corneometry, cutometry, visioscan and ultrasound analyses and (ii) by taking biopsies and subsequent PCR for gene expression analyses related to extracellular matrix homeostasis. Results: Pycnogenol supplementation was well tolerated in all volunteers. Pycnogenol significantly improved hydration and elasticity of skin. These effects were most pronounced in women presenting with dry skin conditions prior to the start of supplementation. The skin-physiological improvement was accompanied by a significant increase in the mRNA expression of hyaluronic acid synthase-1 (HAS-1), an enzyme critically involved in the synthesis of hyaluronic acid, and a noticeable increase in gene expression involved in collagen de novo synthesis. Conclusions: This study provides skin-physiological and for the first time molecular evidence that Pycnogenol supplementation benefits human skin by increasing skin hydration and skin elasticity. These effects are most likely due to an increased synthesis of extracellular matrix molecules such as hyaluronic acid and possibly collagen. Pycnogenol supplementation may thus be useful to counteract the clinical signs of skin aging.

The Effects of Vitamin A Compounds on Hyaluronic Acid Released from Cultured Rabbit Corneal Epithelial Cells and Keratocytes

A role of vitamin A in the synthesis of hyaluronic acid by skin cells is well known. Hyaluronic acid is produced by corneal epithelial cells and keratocytes in the eye. We investigated whether rabbit corneal epithelial cells and keratocytes release hyaluronic acid after exposure to vitamin A compounds. Rabbit corneal epithelial cells and keratocytes were inoculated with RCGM2 medium and incubated at 37ºC under 5% CO(2) in air for 24 h. The medium was then replaced with medium containing 0.1, 1, 10, or 100 μM retinoic acid or retinol palmitate (VApal) and incubated for another 48 h. Hyaluronic acid release from both corneal epithelial cells and keratocytes during culture was increased by retinoic acid at the lower concentration of 0.1 μM and 1 μM determined with a sandwich binding protein assay kit. However, it was significantly decreased at the higher concentrations of 10 μM and 100 μM, and the cell count determined with a Neutral Red assay kit was also decreased at these concentrations. On the other hand, hyaluronic acid release from corneal epithelial cells during culture was increased by VApal at the lower concentration of 0.1 μM and 1 μM, but there was no significant difference in the cell count for either corneal epithelial cells or keratocytes in the presence of VApal at any concentration. In conclusion, it is suggested that vitamin A stimulates the release of hyaluronic acid from cultured rabbit corneal epithelial cells and keratocytes.

Endothelin-1, the Unfolded Protein Response, and Persistent Inflammation

Endothelin-1 is a potent vasoactive peptide that occurs in chronically high levels in humans with pulmonary hypertension and in animal models of the disease. Recently, the unfolded protein response was implicated in a variety of diseases, including pulmonary hypertension. In addition, evidence is increasing for pathological, persistent inflammation in the pathobiology of this disease. We investigated whether endothelin-1 might engage the unfolded protein response and thus link inflammation and the production of hyaluronic acid by pulmonary artery smooth muscle cells. Using immunoblot, real-time PCR, immunofluorescence, and luciferase assays, we found that endothelin-1 induces both a transcriptional and posttranslational activation of the three major arms of the unfolded protein response. The pharmacologic blockade of endothelin A receptors, but not endothelin B receptors, attenuated the observed release, as did a pharmacologic blockade of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) signaling. Using short hairpin RNA and ELISA, we observed that the release by pulmonary artery smooth muscle cells of inflammatory modulators, including hyaluronic acid, is associated with endothelin-1-induced ERK-1/2 phosphorylation and the unfolded protein response. Furthermore, the synthesis of hyaluronic acid induced by endothelin-1 is permissive for persistent THP-1 monocyte binding. These results suggest that endothelin-1, in part because it induces the unfolded protein response in pulmonary artery smooth muscle cells, triggers proinflammatory processes that likely contribute to vascular remodeling in pulmonary hypertension.

Glucosamine Enhances Tissue Regeneration In The Process Of Wound Healing In Rats As Animal Model; A Stereological Study

Introduction: Glucosamine (GA), a water soluble hexosamine made from chitin or chitosan, was showed to have stimulatory effect on matrix formation, enhancing inflammatory response, modulating hyaluronic acid synthesis which promotes extracellular matrix remodeling leading to better wound healing. GA was also shown to have antioxidant and immunomodulatory effect which play roles in wound healing process. In this study we aimed to determine the effect of topical administration of GA on wound healing process in rats as animal model. Material and methods: 36 Wistar rats were divided into 3 groups; the control group which received no treatment; the Glucosamine group that received GA gel with 2% GA concentration, and the Base group that was treated with the vehicle. A 1 cm full-thickness wound was created on the posterior of each rat’s neck. Treatments took place every 24 hours for 15 days. The wound closure rate, volume density of collagen bundles and vessels, fibroblast population, length density and mean diameter of the vessels were estimated by using unbiased stereological and histomorphometrical methods. Results: GA enhanced the wound closure rate consequently as well as fibroblast proliferation, collagen synthesis and proliferation of hair follicles in contrast with the base and the control group. Although not statistically significant, GA also improved the revascularization process in the wound site. Conclusion: Results of this study indicate that GA has the potential for being used for treatment of skin wounds; however, still further evaluations on its mechanisms of action and clinical advantages and disadvantages should be performed.

Rejuvenation of Periorbital Area: Treatment with an Injectable Nonanimal Non-Crosslinked Glycerol Added Hyaluronic Acid Preparation

Therapeutic approaches to aging of the periorbital region are unique because of the delicacy of the anatomical structures and the possibility of adverse events. The synthesis of hyaluronic acid (HA) and other components responsible for skin hydration and elasticity diminish with age. To evaluate the efficacy and safety of an injectable product containing non-crosslinked HA of nonanimal origin in association with glycerol to treat aging of the periorbital region. A pilot study in which 20 women were administered three monthly superficial intradermal injections of non-crosslinked HA of nonanimal origin containing glycerol in the periorbital area. The clinical results consisted of the evaluation of three researchers and an independent evaluator and the degree of posttreatment patient satisfaction. An improvement of between 25% and 50% in skin brightness, texture, and turgor was observed in the periorbital area. Papules were present after each application, and hematoma was the longest lasting effect. All adverse events were reversible and well tolerated. Injections of HA of nonanimal origin, in association with glycerol, using a micropuncture technique are well tolerated and can improve skin brightness and turgor and reduce roughness in the periorbital region.

Effect of adipose derived stem cell-conditioned medium in the expression and synthesis of hyaluronic acid in human dermal fibroblasts

Effect of adipose derived stem cell-conditioned medium in the expression and synthesis of hyaluronic acid in human dermal fibroblasts

Increased HAS2‐driven hyaluronic acid synthesis in shar‐pei dogs with hereditary cutaneous hyaluronosis (mucinosis)

The Chinese shar-pei dog is known for its distinctive feature of wrinkled and thickened skin, defined as primary or hereditary cutaneous mucinosis. In a recent report, we identified the mucinous material deposited in the shar-pei skin as the polysaccharide hyaluronan (HA). In the present work, the molecular and cellular mechanisms underlying this phenotype have been identified in dermal fibroblasts isolated from shar-pei dogs. The production of HA, which appeared to be mainly associated with cell membrane protrusions and also intracellular, was higher in shar-pei fibroblasts than in control cells. The HA accumulation is related to a higher mRNA expression of the isoform HAS2 of the HA-synthesizing enzyme family, hyaluronan synthases (HAS). The higher expression of HAS2 in shar-pei fibroblasts was confirmed at the protein level. The other HAS isoenzymes, HAS1 and HAS3, and the HA-degrading enzymes, Hyal1 and Hyal2, were not differentially expressed in shar-pei fibroblasts compared with cells from control dogs. Fibroblasts from shar-pei dogs and from control dogs are morphologically different as observed by transmission electron microscopy. Scanning electron microscopy revealed a large number of cellular protrusions with associated globular deposits. Electron microscopy after labelling with biotinylated HA-binding protein confirmed an increased HA content in shar-pei fibroblasts, which could be localized in several subcellular structures. The authors propose the name hereditary cutaneous hyaluronosis (HCH) for affected dogs, because it better defines the cutaneous mucinosis of shar-pei dogs.

Collagen Fragments Inhibit Hyaluronan Synthesis in Skin Fibroblasts in Response to Ultraviolet B (UVB): NEW INSIGHTS INTO MECHANISMS OF MATRIX REMODELING

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The α(v)β(3)-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to α(v)β(3) abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by α(v)β(3)-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to α(v)β(3)-blocking antibodies. In conclusion, these data suggest that CF activate α(v)β(3)-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.

Synthesis and properties of hyaluronic acid containing copolymers crosslinked by γ-ray irradiation

Biocompatible and biodegradable hydrogels based on polyvinylpyrrolidone (PVP), gelatin and hyaluronic acid (HA were prepared using a gamma irradiation polymerization technique, and their characteristics were examined. The degree of gelation for these systems was between 55–85% and increased with increasing gelatin or PVP content increased. The gel fraction of the HA/gelatin hydrogel was 30% larger than that of the HA/PVP hydrogel when exposed to a 30–50 kGy radiation dose. The increase in cross-linking density by gamma irradiation was confirmed by TGA. Swelling studies of the hydrogels prepared under various conditions were carried out in aqueous solutions ranging in pH from 2–8 to determine the pH sensitivity. The hydrogels based on HA had the highest swollen water content with a ratio of 1:10 (HA:gelatin and 1:5 (HA:PVP and a 30 kGy radiation dose. HA/gelatin gels showed a large swelling ratio at pH 2 and above pH 5, but the swelling ratio at pH 4 was relatively small. The difference in the swelling degree in various pH media was due to the formation of a polyelectrolyte complex containing oppositely charged polymers. Open image in new window

Abstract 2598: RHAMM- A potential predictor of metastasis and therapeutic target in kidney cancer

Abstract Up to 25% of patients with renal cell carcinoma (RCC) have metastasis at initial diagnosis. Current targeted therapies (e.g., Sorafenib) are not curative and the 5-year survival of these patients is &amp;lt;10%. Molecular determinants of RCC growth and metastasis may be accurate prognostic markers for metastasis and could be targeted for therapy. We evaluated the expression of 5 genes (and their splice variants) as prognostic indicators. We also tested the therapeutic efficacy of the combination of 4-methylumbelliferone (4-MU), a non-toxic, orally bioavailable hyaluronic acid (HA) synthesis inhibitor and Sorafenib (Soraf). Tumor specimens were collected from RCC patients undergoing nephrectomy (n=81; T1, 40; T2+, 41; Grade 1, 5; Grade 2, 32; Grade 3, 25; Grade 4, 19). Nine patients developed metastasis within 24 months. Tissue-RNA was subjected to Q-PCR for HA receptors (CD44, RHAMM), chemokine receptors (CXCR4, CXCR7); and chemokines (SDF1-α,β,γ). mRNA levels were correlated with metastasis by logistic regression. Effect of 4-MU, Soraf and 4-MU+Soraf on cell proliferation, cell cycle and apoptosis was examined in RCC cells (HTB46, 786-O, ACHN, 769-P, A498) by cell counting, flow cytometry and Cell Death ELISA. Boyden chamber was used for motility assay. Effect on cell cycle, apoptosis, and HA receptor levels were evaluated by immunoblotting and Q-PCR. RHAMM expression (157±124) was 8-fold elevated in tumors which later metastasized when compared to those which did not (20.1±31.5; P=0.005). In univariate analysis age, grade, stage and RHAMM levels significantly correlated with metastasis. In multivariate analysis, only RHAMM significantly correlated with metastasis (P=0.03; chi-sq = 4.6; sensitivity 88.9%; specificity 90%). Combination of 4-MU (0.2 mM) and Soraf (2.5 or 5 μM), at non-cytotoxic doses caused 60-90% inhibition in proliferation after 48-72 h treatment and motility in RCC cells. Inhibition of cell proliferation could not be reversed by HA addition. Only the combination induced cycle arrest in late S- and G2-M phases (1.5-2.5-fold) and induced apoptosis (3-4-fold). Cell cycle arrest and apoptosis were confirmed by increased phospho-Chk1 and PARP levels. 4-MU+Soraf combination down regulated CD44 and RHAMM mRNA levels by 2- and 68-fold, respectively. RHAMM expression in RCC tissues is potentially an independent prognostic indicator for metastasis. 4-MU+Soraf combination which targets RHAMM expression inhibits RCC cell growth and motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2598. doi:10.1158/1538-7445.AM2011-2598