Human Skin Fibroblast Cultures Research Articles

Gums as Macromolecular Crowding Agents in Human Skin Fibroblast Cultures.

Even though tissue-engineered medicines are under intense academic, clinical, and commercial investigation, only a handful of products have been commercialised, primarily due to the costs associated with their prolonged manufacturing. While macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition in eukaryotic cell culture, possibly offering a solution in this procrastinating tissue-engineered medicine development, there is still no widely accepted macromolecular crowding agent. With these in mind, we herein assessed the potential of gum Arabic, gum gellan, gum karaya, and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and carrageenan were used as a control). Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (the longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/mL, 75 µg/mL, and 100 µg/mL gum gellan; and the 500 µg/mL and 1000 µg/mL gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrates the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential in the development of tissue-engineered medicines.

Macromolecular crowding in human tenocyte and skin fibroblast cultures: A comparative analysis

Although human tenocytes and dermal fibroblasts have shown promise in tendon engineering, no tissue engineered medicine has been developed due to the prolonged ex vivo time required to develop an implantable device. Considering that macromolecular crowding has the potential to substantially accelerate the development of functional tissue facsimiles, herein we compared human tenocyte and dermal fibroblast behaviour under standard and macromolecular crowding conditions to inform future studies in tendon engineering. Basic cell function analysis made apparent the innocuousness of macromolecular crowding for both cell types. Gene expression analysis of the without macromolecular crowding groups revealed expression of tendon related molecules in human dermal fibroblasts and tenocytes. Protein electrophoresis and immunocytochemistry analyses showed significantly increased and similar deposition of collagen fibres by macromolecular crowding in the two cell types. Proteomics analysis demonstrated great similarities between human tenocyte and dermal fibroblast cultures, as well as the induction of haemostatic, anti-microbial and tissue-protective proteins by macromolecular crowding in both cell populations. Collectively, these data rationalise the use of either human dermal fibroblasts or tenocytes in combination with macromolecular crowding in tendon engineering.

Exploration of SUMO2/3 Expression Levels and Autophagy Process in Fragile X-Associated Tremor/Ataxia Syndrome: Addressing Study Limitations and Insights for Future Research.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in adult FMR1 premutation carriers. The neuropathological hallmark of FXTAS is an intranuclear inclusion in neurons and astrocytes. Nearly 200 different proteins have been identified in FXTAS inclusions, being the small ubiquitin-related modifier 2 (SUMO2), ubiquitin and p62 the most highly abundant. These proteins are components of the protein degradation machinery. This study aimed to characterize SUMO2/3 expression levels and autophagy process in human postmortem brain samples and skin fibroblast cultures from FXTAS patients. Results revealed that FXTAS postmortem brain samples are positive for SUMO2/3 conjugates and supported the idea that SUMO2/3 accumulation is involved in inclusion formation. Insights from RNA-sequencing data indicated that SUMOylation processes are significantly upregulated in FXTAS samples. In addition, the analysis of the autophagy flux showed the accumulation of p62 protein levels and autophagosomes in skin fibroblasts from FXTAS patients. Similarly, gene set analysis evidenced a significant downregulation in gene ontology terms related to autophagy in FXTAS samples. Overall, this study provides new evidence supporting the role of SUMOylation and autophagic processes in the pathogenic mechanisms underlying FXTAS.

Biological Activities of Ceratonia siliqua Pod and Seed Extracts: A Comparative Analysis of Two Cretan Cultivars.

Ceratonia siliqua L., commonly known as the carob tree, appears in most Mediterranean countries, often cultivated for the collection of its fruits to be used as food for humans and animals. This study was aimed at the phytochemical characterization of two common Cretan C. siliqua cultivars and the biological evaluation of deseeded pod and seed extracts regarding their putative use in cosmetics. Gas and liquid chromatographic techniques were used to assess their essential oil, fatty acid, and carbohydrate profiles. Cell-free assays, including free-radical scavenging; the inhibition of tyrosinase and collagenase; the blocking of advanced glycation end product (AGE) formation; along with assays in human skin fibroblast cultures, i.e., reactive oxygen species suppression, glutathione stimulation, and protection from oxidative stress and from ultraviolet (UVB) radiation, were also used. Extracts from both cultivars were found to possess antioxidant capacity, tyrosinase- and collagenase-inhibitory activities, an ability to block glucose-induced AGEs, and in certain cases, UVB absorbance and photoprotective activities. Seed extracts were in general more active, while the use of 30% aqueous methanol seemed to be more efficient than n-hexane for extraction. Serial partition of the most active extracts resulted in fractions with enriched biological activities. These properties make Cretan carob extracts and their fractions suitable candidates for use in cosmetics.

Platelet-rich plasma counteracts interleukin-1 induced inhibition of collagen biosynthesis through recovery of impaired β1-integrin signaling and prolidase activity in human skin fibroblasts.

Although inflammation is the first step in wound healing, it contributes to down-regulation of collagen biosynthesis delaying the process of tissue regeneration. The study was conducted to evaluate the effects of platelet-rich plasma (PRP) on interleukin-1β (IL-1β) – dependent inhibition of collagen synthesis, prolidase activity, and β1-integrin signaling in cultured human skin fibroblasts. PRP was obtain using the SmartPReP®2 Autologous Platelet Concentrate+ System. Collagen biosynthesis and prolidase activity were measured in confluent human skin fibroblast cultures with IL-1β, PRP, hyaluronic acid (HA), and mixtures of IL-1β with PRP or HA. Immunofluorescence bioimaging analysis was employed to evaluate expression of β1 integrin receptor, cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) protein. Incubation of fibroblasts with 2% PRP for 24 h contributed to about 500% increase in collagen biosynthesis and a significant increase in the expression of β1-integrin receptor and prolidase activity, compared to the control cells. In the cells treated with IL-1β, collagen biosynthesis, β1-integrin receptor expression, and prolidase activity were decreased while COX-2 and NF-κB expressions were significantly increased. All these processes were recovered to control values by PRP or HA; however, PRP was found to act more effectively than HA. It was found that PRP counteracted IL-1β -dependent inhibition of collagen synthesis through recovery of β1-integrin receptor expression and prolidase activity and down-regulation of COX-2 and NF-κB expressions in cultured fibroblasts. The data suggest that PRP evoke anti-inflammatory activity that is desirable in tissue regeneration.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10−5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10−34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Impact of <i>Candida spp.</i> metabolites on human skin fibroblasts

Micromycetes spp. have been increasingly involved in the etiology of infectious diseases guiding to consider them not as important as bacterial and viral pathogens. Nowadays, a lot of severe forms of candidiasis are caused by C. auris, C. albicans, whereas C. glabrata and C. krusei are of similar importance. Members of these species were selected to investigate related metabolite action on human skin fibroblasts. Candida spp. being continuously found on the epithelium and mucosal membranes resulting in to sustained interaction between microbiota and human cells. Potential to produce metabolites containing pathogenicity factors is one of the crucial events for transition to invasive candidiasis, wherein human epithelial cells build up the front line of defense barrier preventing Candida spp. invasion into deeper host tissues. The study was aimed at assessing data on metabolite effects derived from epidemiologically relevant Candida spp. on primary human skin fibroblast culture in vitro. In particular, there were analyzed Candida spp. metabolites acting on fibroblast monolayer integrity and viability in cell suspension. It was found that Candida spp. metabolites might directly cause fibroblast death so that biocidal activity was exhibited as a strain-specific feature. A direct biocidity against dermal cells was more typical for strains C. glabrata и C. krusei, less pronounced for C. albicans and very weak for C. auris. In addition, a mechanism for secretory product-related biocidal activity derived from various Candida spp. on dermal fibroblasts in vitro revealed that it resulted in fibroblasts death 1 hour after exposure that peaked at 3 hrs. Cell death was equally proceeded via apoptosis and necrosis. Of note, biocidal effect of fungal metabolites showed no correlation with Candida-related potential to cleave intercellular junctions. It was found that C. auris metabolites showing weak biocidity against some fibroblasts simultaneously resulted in more marked disruption of cell monolayer compared to other Candida spp. Perhaps, it is just a feature of C. auris that might account for its higher invasiveness potential allowing to destroy tight human tissues more effectively compared to other Candida spp.

Biomimetic macroporous hydrogel with a triple-network structure for full-thickness skin regeneration

Full-thickness skin repair is still a challenge in clinical practice because it is extremely difficult for dermal reconstruction and calls for a promising strategy to achieve quick sutureless closure and high-quality dermal restoration for large acute skin defects. Inspired by the unique composition, mechanical properties and microscopic architecture of skin tissue, a novel biomimetic hydrogel with a triple-network structure that mimics the extracellular matrix (ECM) composition has been reported. A composite biomimetic hydrogel was prepared using ECM-derived biopolymers, gelatin methacryloyl (GelMA) and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2‑methoxy-5-nitro-sophenoxy) butanamide (NB)-linked sodium alginate (Alg-NB) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). This biomimetic hydrogel can undergo rapid gelling under UV irradiation (approximately 3 s), has strong mechanical properties (mechanical strength ≈530 kPa) with UV-ionic-crosslinking, and shows strong adhesion to wet tissue. In vitro two-dimensional culture of human skin fibroblasts (HSFs) confirmed that the biomimetic hydrogel has excellent biocompatibility, and HSFs on the surface grow into cell clusters because of the macroporous structures of the hydrogel (pore diameter ≈160 μm). The subcutaneous implantation of the biomimetic hydrogel in Sprague-Dawley (SD) rats confirmed its biocompatibility and low biodegradation in vivo. Moreover, in an SD rat full-thickness skin defect model, this engineered biomimetic hydrogel not only could be used for sutureless wound closure strategies but also could accelerate the reconstitution of dermal tissue with skin appendages. This study provides an effective strategy of quick sutureless wound closure and highly efficient repair for full-thickness skin defects and shows enormous potential for tissue regeneration in clinical applications.

Signaling Molecules of Human Skin Cells as the Targets for Injection Cosmetology.

IntroductionSkin aging is a natural process that cannot be stopped. However, there are many ways to help attenuate premature aging of the skin and reduce the signs that have already appeared. One of them is the subcutaneous administration of preparations containing a combination of hyaluronic acid, active amino acids, and peptides providing an anti-aging clinical effect. The purpose of this research is to study in vitro new signaling molecules with the anti-aging effects and influence of hyaluronic acid fillers on its expression.MethodsThe study was conducted using cell cultures of human facial skin: 1) mixed culture of human facial skin keratinocytes and fibroblasts, and 2) culture of human facial skin keratinocytes enriched with Merkel cells. Immunocytochemistry, confocal microscopy and Western blot were used to identify markers of aging.ResultsHA-Y and HA-S activated the expression of Klotho in the case of aging mixed culture of human skin keratinocytes and Merkel cells. The increase in expression of MTH-1 with aging of cultures provides evidence of activating defense mechanisms against reactive oxygen species that are accumulating with aging, under the action of HA-S and HA-Y. There was a statistically valid increase in the area of expression of melatonin receptor 1A and 1B markers when adding both HA-S and HA-Y to cultured cells.ConclusionThis investigation showed that the studied fillers have biological effects, testifying the stimulation of reparative processes in the skin under their control.

Locked-nucleotide antagonists to varicella zoster virus small non-coding RNA block viral growth and have potential as an anti-viral therapy

Herpes zoster (HZ) remains a significant health burden with millions of cases in North America and Europe annually. HZ is frequently followed by long-term pain or post-herpetic neuralgia (PHN). Although effective vaccines for HZ are available, currently used nucleotide analogues often have limited effectiveness against HZ and especially PHN, so there remains a need for additional antiviral therapies for HZ. We recently identified a population of small non-coding RNA (sncRNA) encoded by Varicella Zoster Virus (VZV) and showed that single locked-nucleic acid antagonists (LNAA) to some sncRNA can modulate VZV replication in cell culture. In this work, we explored the antiviral effects of combinations of LNAA oligonucleotides targeting VZVsncRNA. Combinations of LNAA targeting three VZVsncRNA encoded in and near a critical viral regulatory gene were additive, achieving 96 % reduction in virus growth in a cell line. VZV growth was also inhibited by more than 90 % in primary human skin fibroblast cultures by individual and combinations of LNAA to VZVsncRNA. The inhibition by VZVsncRNA was specific and not a consequence of innate immune responses since LNAA to a different VZVsncRNA enhanced VZV growth. Targeted VZVsncRNA lack homologous sequences in the human transcriptome suggesting that LNAA to them would have reduced cytotoxicity if used as therapeutics. These results support further development of oligonucleotides targeting VZVsncRNA as a novel treatment for HZ.

Antioxidant cerium ions-containing mesoporous bioactive glass ultrasmall nanoparticles: Structural, physico-chemical, catalase-mimic and biological properties

The multifunctional biological properties of Ce ions including antioxidant, anti-inflammatory, antibacterial and anti-cancer effects are very encouraging for development of Ce-containing biomaterials with therapeutic properties. Herein, novel Ce3+/Ce4+ ions containing mesoporous bioactive glass ultrasmall nanoparticles (Ce-BGn) were prepared by a facile one-pot ultrasound-assisted sol-gel method. Interestingly, Ce2O3 incorporation exerted a significant influence on the particle size and textural properties of mesoporous BGn (SiO2 – CaO binary glass system). Ce-BGn exhibited ultrasmall nanoparticle size (< 30 nm), mesoporous texture (pore size up to 2.82 nm and pore volume up to 0.191 cm3/g) and large specific surface area ca. 132.9 m2/g. Notably, in situ formation of CeO2 nanospheres (3–6 nm) was detected at the surface and in the amorphous glass matrix of mesoporous Ce-BGn. Importantly, X-ray photoelectron spectroscopy (XPS) revealed the presence of 72.57 % Ce3+ and 27.43 % Ce4+ at the surface of mesoporous Ce-BGn with Ce3+/Ce4+ ratio = 2.66. Furthermore, mesoporous Ce-BGn exhibited high catalase-mimic activity and showed sustained release of Ce (2.5–32 ppm), Ca (85–327 ppm) and Si (54–200 ppm) ions within 4 weeks along with excellent bone-like hydroxyapatite formation. Finally, the in vitro biological behavior of mesoporous Ce-BGn in cell cultures of human skin fibroblasts (HSF) revealed that mesoporous Ce-BGn (with concentrations up to 300 μg/mL) possess good cyto-biocompatibility. Taken together, novel ultrasmall mesoporous Ce-BGn showed remarkable catalase-mimic activity via surface containing Ce3+/Ce4+ ions which can scavenge ROS (Ce3+↔ Ce4+) and decompose H2O2 molecules into H2O and O2. In addition to that, Ce-BGn demonstrated sustained release of bioactive ions (Ce, Ca and Si), excellent bone-like hydroxyapatite formation and good cyto-biocompatibility.

117-LB: Nonviral Direct Reprogramming of Human Skin Fibroblasts into Hormone-Expressing ß-Like Cell Clusters

While allogenic islet grafting has shown significant promise for the treatment of T1D, donor tissue scarcity and the need for aggressive immunosuppression has limited widespread use of this therapy. Consequently, recent studies have focused on deriving β-like cells from autologoussources (e.g., exocrine pancreas, iPSCs) via reprogramming and/or lengthy differentiation protocols. However, current approaches to β-cell derivation face several hurdles, including limited and/or functionally impaired cell sources, tedious pre-processing steps, and heavy reliance on viral vectors (i.e., for cell transformations), among others. Here we report on the development ofa non-viral approach to β-cell derivation from human skin fibroblasts via direct reprogramming.Human fibroblasts were isolated from surgical discard tissue. Electroporation was then used to screen a pool of transcription factor genes that included Pdx1, Ngn3, MafA, Pax4, and Tcf3, to identify optimum permutations that can drive β-cell-directed conversions in human skin fibroblast cultures. Transfection extent, efficiencies, and transcriptive activity, were evaluated by qRT-PCR,fluorescence microscopy, and RNA sequencing. Transfected fibroblasts were maintained indifferentiation media for ~3 weeks. β-cell-directed transformations were evaluated in terms of morphology and C-peptide expression. Our results indicate that while transfection with Tcf3 alone did not lead to significant changes in cell morphology and phenotype compared to sham, co-transfection of Tcf3 with Pdx1+Ngn3+MafA (PNM) or Pdx1+Ngn3+MafA+Pax4 (PNMP) led to cell clustering, as well as C-peptide expression as early as day 15 post-transfection, which was absent in fibroblasts transfected only with PNM or PNMP. Altogether, our findings indicate that Tcf3 maybe a key factor in enabling direct conversion of human dermal fibroblasts into hormone-expressingβ-like cells for potential applications in β-cell replacement therapies. Disclosure L.R. Lemmerman: None. M. Rincon-Benavides: None. S.A. Tersey: None. B.N. Blackstone: None. H.M. Powell: None. N. Higuita-Castro: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. D. Gallego-Perez: None.

Photochemoprotective effects of Spirulina platensis extract against UVB irradiated human skin fibroblasts

Ultraviolet B (UVB) irradiation is an important cause of a number of harmful side effects such as sunburn, photoaging, pigmentation and skin cancer. Photoprotection, which is used to prevent UVB interaction and to attenuate UVB related damage could be used to prevent the effects of UVB irradiation. Recently, the use of natural products as skin photoprotective agents has been in increasing demand. In this study, we are proposing a panel of in vitro methods for the evaluation of Spirulina platensis extract for its ability to absorb the UVB spectra by determination of its spectrophotometric Solar Protection Factor (SPF), and by attenuating its biological protective effects against UVB-irradiated human skin fibroblast cultures. Our data revealed that S. platensis extract contained high amounts of phenolic compounds (23,465.71 ± 167.72 mg GAE/kg extract) and produced a high degree of absorbance within a wavelength range of 290–320 nm yielding an SPF value of 30.39 ± 3.37 (3 mg/ml), all of which collectively confirmed the photoprotective activity of S. platensis polyphenols. S. platensis extract has no cytotoxicity on UVB irradiation of human skin fibroblasts, peripheral blood mononuclear cells and red blood cells. It was also found to exert antioxidant activities both in vitro and by way of intracellular reactive oxygen species in UVB irradiation fibroblast cells. The induction of interleukin- 6 and -8 secretions by UVB irradiation was dramatically aborted after S. platensis extract treatment. Moreover, the S. platensis extract significantly inhibited tyrosinase activity. Our above results indicated the photochemoprotective effects of S. platensis extract against UVB irradiation in human skin fibroblast cells. Therefore, S. platensis extract could be considered a prospective source of UVB-absorbers with antioxidant capacity. It could also serve as an additive in photoprotective cosmetic formulations which could prevent the harmful effects of UVB radiation.

Гибридные конструкции трикальцийфосфат/гидрогель, предназначенные для регенерации костной ткани и функционализированные противоопухолевым препаратом

Hybrid materials designed for the regeneration of bone defects and consisting of a resorbable ceramic base (tricalcium phosphate, TCP) coated with a layer of hydrophilic biodegradable polymer have been developed. Biocompatibility of the ceramics was evaluated through in vitro tests using cultures of human skin fibroblasts. To increase the therapeutic potential, the created model structures were saturated with the antitumor drug doxorubicin within the composition of the coating of UV-polymerizable hydrogel based on polyacrylamide/polyethylene glycol diacrylate (PAA/PEGDA). The kinetics of drug release was studied by spectrophotometry using saline. The studied hybrid constructs had low cytotoxicity. Saturation of the structures with the antitumor drug resulted in its prolonged release. The results demonstrate the technological feasibility of creating osteoconductive implants based on calcium phosphates suitable for local delivery of antitumor drugs.

The concentration-dependent effect of anethole on collagen, MMP-2 and GAG in human skin fibroblast cultures

PurposeIn aging skin and some skin disorders, components of skin extracellular matrix (ECM) are disturbed and therefore research to find skin drugs is important. Evaluation of anethole impact on collagen, GAGs and MMP-2 in human skin fibroblasts was the aim of this study. Materials and methodsFor collagen assay the Sircol dye, 5-[3H]proline and real time-PCR were used. MMP-2 activity was detected by zymography. GAG concentration was determined using 1,9-dimethylmethylene blue (DMMB). Cell viability was assayed with MTT. ResultsIn cells treated with 1 and 10 μM anethole, a significant increase in collagen synthesis was demonstrated. In contrast, collagen synthesis was significantly decreased in cells exposed to 100 μM anethole. Similar alterations were found in collagen type I expression. The concentration of collagen secreted into the medium was higher only in cells exposed to 1 μM anethole, while it was lower under the influence of higher compound concentrations. It may be due to the lack of pro-MMP-2 activation at 1 μM and a significant increase in the level of MMP-2 at 10 and 100 μM anethole. GAG concentration was reduced under the influence of 100 μM anethole, whereas anethole at lower concentrations revealed the ability to prevent H2O2-induced GAG increase. No significant cytotoxicity of anethole to fibroblasts was noted. ConclusionsOur findings demonstrate the concentration-dependent action of anethole on the crucial components of ECM in cultured skin fibroblasts, which may be somewhat beneficial and may possibly be developed towards a therapeutic use in some skin disorders.

A MicroRNA-29 Mimic (Remlarsen) Represses Extracellular Matrix Expression and Fibroplasia in the Skin

MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.

A correlation of long term effects and radiation quality in the progeny of bystander cells after microbeam radiations: The experimental study of radiotherapy for cancer risk mitigation

The goal of this study is to investigate the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of delayed stressful effects in the progeny of bystander human skin fibroblasts cultures (NB1RGB). Briefly, confluent NB1RGB cells in the presence and absence of gap junction inhibitor (AGA) were exposed to ionizing radiation (IR) with a different linear energy transfer (LET) either 5.35 keV X rays (LET ∼6 keV/μm) or 18.3 MeV/u carbon (LET ∼103 keV/μm) microbeam radiations. Following 20 populations post-irradiation, the progeny of bystander NB1RGB cells were harvested and assayed for several of biological endpoints. Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to low-LET X rays showed the persistence of oxidative stress and it was correlated with the increased mutant fraction. Such effect were not observed after high-LET carbon ions. Interestingly, inhibition of GJIC mitigated the toxic effects in the progeny of bystander cells. Together, the results contribute to the understanding of the fundamental radiation biology relating to the high-LET carbon ions to mitigate cancer risk after radiotherapy. Furthermore, GJIC be considered as a critical mediator in the bystander mutagenic effect.

Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

AimThe aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes.Materials and methodsStudies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression.ResultsDepending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol.ConclusionThis study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells.

Objective: Laryngeal squamous cell carcinoma is one of the most common malignant tumors in the head and neck region. Due to the poor response to chemotherapeutics in patients and low survival rate, successful treatment of larynx cancer still remains a challenge. Therefore, the identification of novel treatment options is needed. We investigated the anticancer effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on two different laryngeal cancer cell lines RK33 and RK45. We also studied the antiproliferative action of SAHA in combination with cisplatin and defined the type of pharmacological interaction between these drugs. Materials and Methods: Viability and proliferation of larynx cancer cell lines were studied by methylthiazolyldiphenyl-tetrazolium bromide method and 5-bromo-2-deoxyuridine incorporation assay, respectively. The type of interaction between SAHA and cisplatin was determined by an isobolographic analysis. Western blotting, flow cytometry and quantitative polymerase chain reaction method were used to determine acetylation of histone H3, cell cycle progression and genes expression, respectively. Apoptosis was assessed by means of nucleosomes released to cytosol. Results: SAHA alone or in combination with cisplatin inhibited larynx cancer cells proliferation, whereas displayed relatively low toxicity against normal cells - primary cultures of human skin fibroblasts. The mixture of SAHA with cisplatin exerted additive and synergistic interaction in RK33 and RK45 cells, respectively. We showed that SAHA induced hyperacetylation of histone H3 K9, K14 and K23 and triggered apoptosis. SAHA also caused cell cycle arrest by upregulation of CDKN1A and downregulation of CCND1 encoding p21WAF1/CIP1 and cyclin D1 proteins, respectively. Conclusion: Our studies demonstrated that SAHA may be considered as a potential therapeutic agent against larynx tumors.

Growth Suppressing Activity of Lhx2 in Human T Cell Acute Lymphoblastic Leukemia (T-ALL)-Derived Cells and Large-Scale Screening of a Lead Compounds Targeting T-ALL

Acute lymphoblastic leukemia (ALL) is one of the most frequently occurring cancers in infants and young children. For patients suffering from CD20-positive B-cell ALL (B-ALL) and Ph-positive ALL, overall survival rates have been greatly improved after clinical introduction of rituximab and imatinib, respectively. However, T-cell ALL (T-ALL) patients still exhibit poor prognosis, since there is no such an efficient molecular-targeted drug.It is known that LIM-only transcriptional co-factor LMO2 and its target gene HHEX are essential for self-renewal of T cell precursors and onset of T-ALL. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related downstream genes. Recently, we reported that overexpression of LIM-homeodomain transcription factor Lhx2 results in liberation of Lmo2 protein from the Lmo2-Ldb1 complex followed by degradation by ubiquitin-proteasome system. We found that proliferation of 5 different human T-ALL-derived cell lines including CCRF-CEM was significantly suppressed by retroviral overexpression of Lhx2. In contrast, enforced overexpression of Lhx2 did not reduce the growth rate of B lymphoma-derived cell line Raji, oral cancer-derived cell line HSC-3, and osteosarcoma-derived cell line Saos-2. Majority of the Lhx2-transduced CCRF-CEM cells was arrested in G0 phase and subsequently underwent apoptosis. Expression of LMO2 protein and HHEX mRNA was repressed by the Lhx2 transduction. Importantly, the Lhx2-mediated growth inhibition was partially rescued by the simultaneous overexpression of Lmo2. However, both C-terminal LIM-domain and homeodomain of Lhx2 were required for the growth-suppressive activity. These data indicated that Lhx2 is capable to blocking proliferation of T-ALL-derived cells in LMO2-dependent and independent fashions. Lhx2 would be a useful molecular tool for designing a new type of anti-T-ALL drug.In order to develop a new drug that mimics the aforementioned activity of Lhx2, we performed large-scale screening of natural compound libraries to find out a compound that suppresses the proliferation of T-ALL cell line CCRF-CEM, but does not inhibit the growth of B lymphoma cell line Raji. Among 150,000 different compounds, we successfully identified 3 low-molecular-weight compounds (44D-L008, 31D-F005, 21D-D016) that fulfilled the above criteria. 44D-L008 and 31D-F005 possessed a common chemical structure. In the presence of 5μM of 44D-L008 and 31D-F005, proliferation of 5 human T-ALL cell lines including CCRF-CEM was severely blocked. On the other hand, Raji, HSC-3 and Saos-2 were completely resistant to both compounds in the same experimental settings. Intriguingly, 44D-L008 decreased viability of human skin fibroblasts in culture, whereas 31D-F005 displayed no such negative effects on skin fibroblasts, peripheral blood mononuclear cells, and peripheral blood T cells of human origin. These results indicated that small differences in the chemical structure between 44D-L008 and 31D-F005 are responsible for the side effect on normal cells. Finally, we collaborated with a hospital and found that 0.5 μM of 31D-F005 efficiently suppressed the in vitro growth of primary cancer cells of a T-ALL patient. Taken together, theses data demonstrated that 31D-F005 is a promising lead compound for a new anti-T-ALL drug. DisclosuresNo relevant conflicts of interest to declare.