Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis.
All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.
Wound dressings with silver have been shown to be cytotoxic in vitro. However, the extrapolation of this cytotoxicity to clinical settings is unclear. We applied dressings with various forms of silver on porcine skin ex vivo and investigated silver penetration and DNA damage. We assessed antimicrobial efficacy, cytotoxicity to skin cells, and immune response induced by the dressings. All dressings elevated the DNA damage marker γ-H2AX and the expression of stress-related genes in explanted skin relative to control. This corresponded with the amount of silver in the skin. The dressings reduced viability, induced oxidative stress and DNA damage in skin cells, and induced the production of pro-inflammatory IL-6 by monocytes. The oxidative burst and viability of activated neutrophils decreased. The amount of silver released into the culture medium varied among the dressings and correlated with in vitro toxicity. However, antimicrobial efficiencies did not correlate strongly with the amount of silver released from the dressings. Antimicrobial efficiency and toxicity are driven by the form of silver and the construction of dressings and not only by the silver concentration. The damaging effects of silver dressings in ex vivo skin highlight the importance of thorough in vivo investigation of silver dressing toxicity.
- MeSH
- buněčné linie MeSH
- infekce v ráně MeSH
- kůže chemie cytologie účinky léků MeSH
- lidé MeSH
- obvazy škodlivé účinky MeSH
- poškození DNA * MeSH
- prasata MeSH
- stříbro toxicita MeSH
- techniky tkáňových kultur MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW.
- MeSH
- buněčná imunita účinky léků genetika MeSH
- cyklooxygenasa 2 genetika MeSH
- kur domácí MeSH
- kyselina hyaluronová farmakologie MeSH
- makrofágy účinky léků imunologie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- oxid dusnatý genetika MeSH
- RAW 264.7 buňky MeSH
- regulace genové exprese účinky léků MeSH
- Streptococcus equi chemie MeSH
- synthasa oxidu dusnatého genetika MeSH
- TNF-alfa genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.
- MeSH
- antigeny CD44 metabolismus MeSH
- chemokiny biosyntéza genetika MeSH
- fibroblasty účinky léků imunologie MeSH
- interleukin-6 biosyntéza genetika metabolismus MeSH
- interleukin-8 biosyntéza MeSH
- kyselina hyaluronová farmakologie MeSH
- leukocyty účinky léků metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- mediátory zánětu metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- molekulová hmotnost MeSH
- přirozená imunita účinky léků genetika MeSH
- signální transdukce účinky léků genetika MeSH
- škára cytologie MeSH
- TNF-alfa biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (Mw=15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelle's interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX.
- MeSH
- antitumorózní látky fytogenní aplikace a dávkování chemie MeSH
- fibroblasty účinky léků MeSH
- HCT116 buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- kultivované buňky MeSH
- kyselina hyaluronová aplikace a dávkování chemie MeSH
- lidé MeSH
- micely * MeSH
- nosiče léků aplikace a dávkování chemie MeSH
- paclitaxel aplikace a dávkování chemie MeSH
- polymery aplikace a dávkování chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.
- MeSH
- antigeny CD44 biosyntéza genetika MeSH
- exprese genu MeSH
- glukuronosyltransferasa biosyntéza genetika MeSH
- hyaluronoglukosaminidasa biosyntéza genetika MeSH
- interleukin-6 biosyntéza MeSH
- interleukin-8 biosyntéza MeSH
- keratinocyty účinky léků metabolismus účinky záření MeSH
- kůže účinky léků metabolismus účinky záření MeSH
- kyselina hyaluronová metabolismus farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- toll-like receptor 2 biosyntéza genetika MeSH
- transformující růstový faktor beta biosyntéza MeSH
- ultrafialové záření MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The regulatory functions of glycosaminoglycan hyaluronan (HA) are suggested to be dependent on its molecular weight (MW). Proinflammatory and stimulatory effects are proposed mainly for the low MW HA. However, the complex response of blood phagocytes to HA of different MW is unclear. Herein, the effects of highly purified HA of precisely defined MW (52, 250, and 970 kDa) on human blood phagocytes were tested. All MW HA activated blood phagocytes, including the spontaneous production of ROS, degranulation, and the production of tumor necrosis factor alpha, with low MW HA 52 kDa having the highest potency and high MW HA 970 kDa having the lowest potency. Interestingly, HA inhibited ROS production stimulated by opsonized zymosan particles and, in contrast, potentiated starch-activated ROS production, mostly independent of MW. Data showed a significant effect of HA of different MW on blood phagocytes, including high MW HA.
- MeSH
- antigeny CD11b krev MeSH
- CD antigeny krev MeSH
- degranulace buněk účinky léků MeSH
- fagocyty účinky léků fyziologie MeSH
- GPI-vázané proteiny krev MeSH
- kyselina hyaluronová chemie farmakologie MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- molekuly buněčné adheze krev MeSH
- reaktivní formy kyslíku krev MeSH
- receptory komplementu 3b krev MeSH
- TNF-alfa krev MeSH
- zánět krev etiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- techniky in vitro MeSH
OBJECTIVES: Hyaluronan, a linear glycosaminoglycan, is an abundant component of extracellular matrix. In its native form, the high-molar-mass hyaluronan polymers have an array of structural and regulatory, mainly anti-inflammatory and anti-angiogenic, functions. In contradiction, the biological effects of fragmented low molecular weight hyaluronan are suggested to be pro-angiogenic and pro-inflammatory. METHODS: The effects of highly purified pharmacological grade hyaluronan of defined molecular weights 11, 52, 87, 250 and 970 kilodaltons were tested on mouse macrophage cell lines RAW 264.7 and MHS. The surface expression of CD44 and Toll-like receptor 2, surface receptors for hyaluronan, was determined by flow cytometry. Activation of macrophages was determined based on nitric oxide and tumour necrosis factor alpha production, inducible nitric oxide synthase expression, and the activation of the nuclear factor kappa B transcriptional factor. RESULTS: Both macrophage cell lines expressed CD44 and Toll-like receptor 2, which were significantly increased by the pre-treatment of macrophages with bacterial lipopolysaccharide. Hyaluronan of any molecular weight did not activate production of nitric oxide or tumour necrosis factor alpha in any mouse macrophage cell lines. Correspondingly, hyaluronan of any tested molecular weight did not stimulate nuclear factor kappa B activation. Similarly, hyaluronan of any molecular weight neither exerted stimulatory nor inhibitory effects on macrophages pre-treated by lipopolysaccharide. CONCLUSION: Interestingly, the data does not support the current view of low molecular weight hyaluronan as a pro-inflammatory mediator for macrophages. Further studies are necessary to clarify the effects of different molecular weight hyaluronan on phagocytes.
- MeSH
- aktivace makrofágů účinky léků fyziologie MeSH
- antigeny CD44 metabolismus MeSH
- buněčné linie MeSH
- kyselina hyaluronová chemie metabolismus farmakologie MeSH
- lipopolysacharidy toxicita MeSH
- makrofágy chemie účinky léků fyziologie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- NF-kappa B metabolismus MeSH
- oxid dusnatý metabolismus MeSH
- průtoková cytometrie MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- TNF-alfa metabolismus MeSH
- toll-like receptor 2 metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
