Search

BACKGROUND: Some scholars have found that dermal papilla spheroid–derived exosomes could promote the development of hair follicles. However, whether adipose-derived stem cell exosomes (ADSC-Exos) have a similar effect on hair growth has not been determined yet. Thus, the purpose of this article was to detect whether ADSC-Exos could promote hair regeneration. METHODS: Adipose-derived stem cells (ADSCs) were isolated from 6-week-old C57BL/6 mice. Then, ADSC-Exos were isolated from the ADSCs. Western blotting was used to detect specific exosome markers. The particle size and distribution of the exosomes were analyzed by NanoSight dynamic light scattering. A total of 12 nude mice were randomly divided into two groups (n = 6 each): the ADSC-Exos group and the control group. For the control group, a mixture of freshly isolated dermal cells (DCs) and epidermal cells (ECs) was grafted. For the ADSC-Exos group, a mixture of DCs, ECs, and 50 μg/ml of ADSC-Exos was grafted. Gross evaluation of the hair regeneration was carried out 2–3 weeks after the transplantation, and the graft site was harvested for histology at the third week. RESULTS: The existence of exosomes derived from ADSCs was evidenced by CD63, ALX1, and CD9 expression. Two or three weeks after the grafting, the number of regenerated hairs in the ADSC-Exos group was higher than that in the control group (p

Keloid scars are associated with physical and psychological sequelae. No studies have investigated the general public's understanding of keloids. Targeted, short educational interventions in susceptible individuals may aid understanding of the condition and compliance with treatment. We aimed to identify the population with the highest prevalence and lowest knowledge.We surveyed four countries to determine the public's understanding of keloids. A quantitative, subjective and cross-sectional street survey was designed using the knowledge, attitudes and practice model principles. The target populations were cities in Ghana, Australia, Canada and England. Surveyors used a hybrid stratified/convenience sampling method. Primary outcomes were prevalence, exposure to keloids as an entity and overall keloid knowledge score compared across demographic groups. Study data have been made fully available for reproducibility and education (https://doi.org/10.17605/OSF.IO/3KZ5E).There were 402 respondents, with a median age of 32 (interquartile range 25-45.25) years, of which 193 were females. The survey was carried out between June 2015 and October 2017. The prevalence of self-identified keloids was 11% in Ghana, 6% in Australia, 2% in Canada and 7% in England. Prevalence, exposure and knowledge were higher in the Ghanaian population.There was association between knowledge, prevalence and the exposure to keloids as an entity. Findings may suggest targeting public health campaigns towards populations where knowledge is lowest, and exposure to and prevalence of keloids are the highest.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive respiratory disease. Arguably, the complex interplay between immune cell subsets, coupled with an incomplete understanding in disease pathophysiology, have contributed to the paucity of successful therapies. In turn, IPF remains a fatal disease at present. Thus, with no effective therapy for either the prevention or treatment of IPF, the need for new therapies is paramount. Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of IPF. Methods:The aim of this study was to test the therapeutic effect of adipose-derived mesenchymal stem cells in a bleomycin-induced pulmonary fibrosis model. First, MSC cells were obtained from mice, and then flow cytometry and cell differentiation culture methods were uesd to identify them. Then adult C57BL/6 mice were challenged with endotracheal instillation of bleomycin and treated with MSC concurrently for reversal models at day 14. Experimental groups were assessed at day 14, 21, or 28. Finally, lung fibroblasts were challenged with TGF-β1 and treated with MSC supernatant or MSCs to explore the relevant mechanisms of pulmonary fibrosis reversal. Results:We successfully obtained mesenchymal stem cells from mouse adipose tissue and identified their differentiation ability and cell phenotype.We found that the presence of MSC or its supernatant could promote the proliferation and migration of lung fibrotic cells. Meanwhile, MSC supernatant could reduce lung collagen deposition, improve Ashcroft score, and reduce the gene and protein content of lung fibrosis-related substances. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively reversed by MSC treatment.Conclusions: In summary, this study found that MSC therapy can prevent and revert the core features of bleomycin-induced pulmonary fibrosis, and the beneficial effects of MSC may be mediated via its paracrine-related cytokines. It provides further evidence for the importance of the MSC in cell therapy medicine applications.

Background Regenerative Epithelial Suspension can lead to the restoration of wound and repigmentation, which can be gained by ReCell medical device to treat scar and depigmentation diseases. Objectives To report the effectivity of ReCell combined with microdermabrasion in scar and depigmentation diseases and review the literature of this new technology. Methods We gave a differential donor⁄recipient ratio of about 1:20-30 with vitiligo, 1:40 with postburn construction, 1:80 with acne scar, and 1:120 with adult congenital melanocytic nevus, 1:80 with pediatrics, respectively. Photographs of patients before treatment and 3 months following the last treatment session were used to evaluate the effectivity. Results A total of 8 patients including vitiligo vulgaris, postburn reconstruction, acne scars, and congenital melanocytic nevi treated by ReCell technology combined with microdermabrasion showed significant improvement in skin texture and color. And 17 studies of the research on ReCell technology were totally included in the systematic review. Conclusion Our investigation showed that Regenerative Epithelial Suspension gained by ReCell technology combined with microdermabrasion may improve scar and depigmentation diseases.

Full-thickness skin regeneration is still a clinical challenge for skin defects. Porcine small intestinal submucosa (SIS) has been exploited as a new scaffold for tissue reconstruction due to its excellent biocompatibility and ease of handling and modification. However, the application of SIS is dramatically impeded by its compact structure. Thus, a strategy for improving this property of SIS is highly desirable. Herein, SIS was recross-linked by a four-arm polyethylene glycol (fa-PEG) with succinimidyl glutarate-terminated branches into a three-dimensional (3D) bioactive sponge (SIS-PEG), which possessed porous 3D frameworks to mimic the structure of skin. The addition of a suitable proportion of fa-PEG endowed SIS with a uniform pore size, outstanding bioactivity, and flexible shape to promote a rapid healing of a mouse skin defect. Compared with SIS, the bioactive SIS-PEG sponge exhibited excellent mechanical stability and was less prone to collagenase degradation. Moreover, SIS-PEG provided a minimally invasive way to deliver stem cells for in situ wound repair. Remarkably, in vivo evaluation demonstrated that dissociated epidermal and dermal cells loaded with SIS-PEG could form reconstituted skin with regenerated hair after 21 days of treatment. The SIS-PEG bioactive sponge exhibited great potential for skin tissue engineering.

Adipose-derived stem cell (ADSC) transplantation has emerged as a potential tool for the treatment of cardiovascular disease and skin wounds. However, with a limited renewal capacity and the need for mass cells during the engraftment, strategies are needed to enhance ADSC proliferative capacity. In this study, we explored the effects of Exendin-4, a glucagon-like peptide-1 analog, on the growth of ADSCs, focusing in particular on phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) and Wnt signaling pathways. Firstly, ADSCs were isolated and cultured in vitro. Then, flow cytometry demonstrated that ADSCs were positive for CD44, CD90 and CD29 but negative for CD31, CD34, and CD45. Exendin-4 (0–200 nM) treatment increased ADSC proliferation. In order to examine specific signaling pathways, a western blotting assay was performed. Our results demonstrate that after treated with 50 nM Exendin-4 for 48 h, the phosphorylation of PI3K, Akt, and GSK3β were increased and phosphorylation of β-catenin was decreased. From these results, we concluded that PI3K-Akt and Wnt-β-catenin signaling pathways mediate Exendin-4 induced ADSC proliferation, the function of which might contribute to the regulation of ADSC proliferation. Our findings provided new insights into the function of the mechanisms underlying Exendin-4 of ADSCs.

Background Human adipose tissue-derived mesenchymal stem cells (ASCs) have potential utility as modulators of the regeneration of tissue that is inflamed or scarred secondary to injuries such as burns or trauma. However, the effect of ASCs on one particular type of scarring, keloidal disease, remains unknown. The absence of an optimal model for investigation has hindered the development of an effective therapy using ASCs for keloids. Objective To investigate the influence of ASCs on angiogenesis, extracellular matrix deposition, and inflammatory cell influx in keloids. Methods We analyzed the proliferation, migration, and apoptosis of human keloid-derived fibroblasts treated with a starvation-induced, conditioned medium from ASCs (ASCs-CM). This was achieved by Brdu proliferation assay, a validated co-culture migration assay, and flow cytometry, respectively. To assess the change in phenotype to a pro-fibrotic state, fibroblasts were analyzed by real-time PCR and contraction assay. A keloid implantation animal model was used to assess the paracrine effect of ASCs histochemically and immunohistochemically on scar morphology, collagen deposition, inflammatory cell composition, and blood vessel density. In tandem, an antibody-based array was used to identify protein concentration in the presence of ASCs-CM at time point 0, 24, and 48 h. Results ASCs-CM inhibited the proliferation and collagen synthesis of human keloid-derived fibroblasts. ASCs-CM was associated with reduced inflammation and fibrosis in the keloid implantation model. Thirty-four cytokines were differentially regulated by ASCs-CM at 24 h. These included molecules associated with apoptosis, matrix metalloproteases, and their inhibitors. The same molecules were present at relatively higher concentrations at the 48 h timepoint. Conclusion These results suggest that ASCs are associated with the inhibition of fibrosis in keloids by a paracrine effect. This phenomenon may have utility as a therapeutic approach in the clinical environment.

Background: Platelet-rich plasma (PRP) is a portion of blood plasma enriched with platelets widely investigated for accelerating bone and soft tissue healing. The purpose of this study is to evaluate the effects of PRP on the previous established photoaging model in vitro and to elucidate the potential anti-photoaging mechanisms of PRP. Methods: Murine dermal fibroblasts (MDFs) were isolated and cultured with or without PRP after exposure to repeated UVB irradiation per protocol. The senescent phenotypes were compared among groups, including cell morphology, senescence-associated β-galactosidase (SA-β-gal) expression, cell cycle arrest, production and degradation of extracellular matrix (ECM), ROS generation, and the alteration of major intracellular antioxidants. Results: 1% PRP effectively reduced the amount of flattened cells and significantly decreased the staining intensity as well as the percentage of SA-β-gal-positive cells when compared with UVB group. Furthermore, PRP appeared to prevent cell cycle arrest caused by irradiation by decreasing p53 and p21 expression. The PRP had demonstrated direct effects on ECM, including increase in type I collagen production and decrease in MMPs expression. Additionally, the increase of glutathione, one of the major intracellular antioxidants, induced by PRP provided a possible explanation for its therapeutic effect. Conclusions: Our work confirmed that PRP, a compound of various growth factors, could counteract senescence-like phenotypes at the cellular level, mirroring its rising clinical popularity in anti-aging and regenerative medicine. The promising effects of PRP calls for future study for further exploration of the underlying mechanisms, which will lead to a broadened application of PRP in future clinical use.

GNB2L1 is an intercellular scaffold protein of the Trp-Asp (WD) repeat protein family, and has been reported to play suppressive roles in the progression of gastric cancer. However, the regulatory mechanisms of GNB2L1 in gastric cancer still remain largely elusive. In the present study, we found that OGT was capable to interact with GNB2L1 directly and modify GNB2L1 with O-GlcNAcylation in gastric cancer, and this O-GlcNAcylation hindered the inhibition of GNB2L1 on migration of gastric cancer cells. Moreover, O-GlcNAcylation regulated the degradation instead of the synthesis of GNB2L1 in gastric cancer, and our data suggested the O-GlcNAcylation on GNB2L1 influenced its stability directly. In addition, the clinical data revealed the negative correlation of the protein level instead of the mRNA level of GNB2L1 with OGT expression, and showed that OGT reversed the inhibition of GNB2L1 on metastasis, and worsened the prognosis of GNB2L1(High) patients. In summary, this study indicated the O-GlcNAcylation on GNB2L1 reversed its inhibition on gastric tumour metastasis via promoting its degradation.

This study provides a novel method in injectable tissue engineering which contains porcine extracellular matrix (ECM) powder scaffolds and stromal-vascular fraction (SVF) cells. We fabricated ECM powders from porcine adipose tissue and mixed the SVF cell suspension together with the ECM powders immediately after the isolation. SVF-loaded ECM powders and their controls were respectively injected into the dorsa of mice. After 6 weeks, the interest sites were harvested and analyzed using histology, immunohistochemistry, and quantitative-polymerase chain reaction (qPCR). Histology and immunohistochemistry demonstrated that the presence of the SVF cells loaded in the ECM powders promoted adipogenesis, angiopoiesis and matrix remodeling. This was in parallel with upregulation of host cell recruitment and adipogenesis genes. The results provide cellular and molecular evidence suggesting a novel method in injectable tissue engineering. We believe that SVF-loaded ECM powders could act as efficient injectable biomaterials for tissue engineering and have great potential for meeting clinical challenges in regenerative medicine, particularly in relation to adipose tissue engineering.

Hypertrophic scar (HPS) is a manifestation of abnormal tissue repair, representing excessive extracellular matrix production and abnormal function of fibroblasts, for which no satisfactory treatment is available at present. Here we identified a natural product of flavonoid, dihydromyricetin, could effectively attenuate HPS formation. We showed that local intradermal injection of dihydromyricetin (50 μM) reduced the gross scar area, cross-sectional size of the scar and the scar elevation index in a mechanical load-induced mouse model. In addition, dihydromyricetin treatment also markedly decreased collagen density of the scar tissue. Furthermore, both in vitro and in vivo study both demonstrated that dihydromyricetin inhibited the proliferation, activation, contractile and migration abilities of hypertrophic scar-derived fibroblasts (HSFs) but did not affect HSFs apoptosis. Western blot analysis revealed that dihydromyricetin could down-regulate the phosphorylation of Smad2 and Smad3 of TGF-β signaling. Such bioactivity of dihydromyricetin may result from its selective binding to the catalytic region of activin receptor-like kinase 5 (ALK5), as suggested by the molecular docking study and kinase binding assay (12.26 μM). Above all, dihydromyricetin may prove to be a promising agent for the treatment of HPS and other fibroproliferative disorders.

Background Rosiglitazone (RO), a second-generation thiazolidinedione used mainly in the treatment of non-insulin-dependent diabetes mellitus, has been discovered to be a high-affinity ligand for peroxisome proliferator-activated receptor-γ (PPAR-γ). Several studies have revealed that PPAR-γ is also involved in the regulation of oxidative stress and chronic inflammation associated with aging process in vivo as well as with cellular senescence in vitro. We sought to investigate whether RO pretreatment will counteract the photoaging process using a well-established cellular photoaging model. Methods Murine dermal fibroblasts (MDFs) were cultured in the absence or presence of RO for 48 h, followed by exposure to repeated UVB irradiation. The senescent phenotypes were evaluated including cell viability, senescence-associated β-galactosidase (SA-β-gal) expression, cell morphology, ROS generation, cell cycle, production and degradation of extracellular matrix (ECM), and the potential mechanisms were discussed. Results Pretreatment with RO (40 μM) significantly decreased the staining intensity and the percentage of SA-β-gal-positive cells and reserved the elongated cell shape compared with UVB group. The cells pretreated with RO also showed decreased UVB-induced degradation of type I collagen by decreasing MMPs expressions. In addition, we observed counteraction of cell-cycle arrest and repression of UVB-induced p53 and p21 in the presence of RO. We further confirmed a significant decrease in ROS accumulation accompanied by an increase in catalase in RO group. Conclusions RO, a potent PPAR-γ activator, counteracts senescence-like phenotypes, including long-term growth arrest, flattened morphology, degradation of ECM and SA-β-gal-positive staining in MDFs by inhibiting the expression of MMPs and increasing the synthesis of catalase when administered to repeated UVB irradiation. The novel application of RO may lead to innovative and effective anti-photoaging therapies.

GNB2L1 and its O-GlcNAcylation has been reported to play roles in gastric cancer metastasis. However, the roles of GNB2L1 in chemoresistance of gastric cancer has never been determined. In the present study, we found that GNB2L1 was downregulated in chemoresistant patients of gastric cancer, and observed the decrease of GNB2L1 in protein levels instead of mRNA levels in different chemoresistant gastric cancer cell lines. Further we proved that this downregulation of GNB2L1 was resulted from its elevated O-GlcNAcylation catalyzed by OGT in both cell lines and patients. Next, we investigate the function of GNB2L1 and its O-GlcNAcylation on gastric cancer metastasis during chemoresistance, and confirmed Ser124 as the major O-GlcNAcylation site on GNB2L1 that regulated its function on metastasis. Furthermore, our data demonstrated that GNB2L1 modulated EMT via regulating the translation of EMT-related proteins in the process of chemoresistance. In summary, this study indicated that GNB2L1 and its O-GlcNAcylation regulated metastasis via modulating the translation of EMT-related proteins in the chemoresistance of gastric cancer.

Background/Aims: Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin photoaging. Murine dermal fibroblasts (MDFs) were subjected to a series of 4 sub-cytotoxic UVB doses (120 mJ/cm2), resulting in changes in cell shape, DNA damage, cell cycle arrest, extracellular matrix variations, reactive oxygen species (ROS) generation, and alterations in major intracellular antioxidant and cellular autophagy levels. Rapamycin (RAPA) is a new macrolide immunosuppressive agent that is primarily used in oncology, cardiology, and transplantation medicine and has been found to extend the lifespan of genetically heterogeneous mice. Several studies have shown that RAPA may have anti-aging effects in cells and organisms. Thus, in this study, we explored the effects and mechanisms of RAPA against the photoaging process using a well-established cellular photoaging model. Methods: We developed a stress-induced premature senescence (SIPS) model through repeated exposure of MDFs to ultraviolet B (UVB) irradiation. The cells were cultured in the absence or presence of RAPA for 48 h. Senescent phenotypes were assessed by examining cell viability, cell morphology, senescence-associated β-galactosidase (SA-β-gal) expression, cell cycle progression, intracellular ROS production, matrix metalloproteinase (MMP) synthesis and degradation, extracellular matrix (ECM) component protein expression, alterations in major intracellular antioxidant levels, and the cellular autophagy level. Results: Compared with the UVB group, pretreatment with RAPA (5 µM) significantly decreased the staining intensity and percentage of SA-β-gal-positive cells and preserved the elongated cell shape. Moreover, cells pretreated with RAPA showed inhibition of the reduction in the type I collagen content by blocking the UVB-induced upregulation of MMP expression. RAPA also decreased photoaging cell cycle arrest and downregulated p53 and p21 expression. RAPA application significantly attenuated irradiation-induced ROS release by modulating intracellular antioxidants and increasing the autophagy level. Conclusions: Our study demonstrated that RAPA elicited oxidative damage in vitro by reducing ROS accumulation in photoaged fibroblasts. The anti-aging effect can be attributed to the maintenance of normal antioxidant and cellular autophagy levels. However, determination of the definitive mechanism requires further study.

UVA1 phototherapy, a new therapeutic approach, has recently been shown good efficacy in the treatment of palmoplantar pustulosis (PPP). The purpose of this study was to compare the efficacy of UVA1 and narrowband UVB (NB-UVB) therapy in the treatment of PPP. Patients with PPP were randomly assigned to either UVA1 or NB-UVB therapy according to a left-right randomization table. Both treatments were performed three times weekly for up to 30 sessions. Clinical evaluation was based on the Palmoplantar Pustular Psoriasis Area and Severity Index (PPPASI) score. Totally 64 patients completed the study. Both UVA1 and NB-UVB therapy showed a statistically significant reduction of PPPASI score compared with the baseline value at the end of the treatment period (P

The commercial use of epidermal growth factor (EGF) is extensive and has been shown to be effective for skin wound healing in clinical practice. There is evidence to indicate that the topical administration of EGF significantly accelerates re-epithelialization by promoting keratinocyte mitogenesis and migration following acute injury; however, the mechanisms involved remain to be elucidated. Thus, in this study, we focused on Kindlin-1, a four-point-one, ezrin, radixin, moesin (FERM)-domain-containing adaptor protein, and report its contribution to EGF-induced re-epithelialization in skin wound healing. In tissue samples, the expression of Kindlin-1 was induced upon EGF treatment compared to that in the natural healing group. In immortalized human keratinocytes (HaCaT cells), we further proved that Kindlin-1 was necessary for mediating EGF-induced activation signals, including integrin β1 activation, focal adhesion kinase (FAK) phosphorylation and actin re-organization, which finally led to enhanced cell proliferation and migration. These results indicate that Kindlin-1 is essential in EGF-induced re-epithelialization in skin wound healing and provide additional rationale for the clinical application of EGF in the treatment of acute wounds.

Background Histone deacetylase (HDAC) inhibitors are widely used in clinical investigation as novel drug targets. For example, panobinostat and vorinostat have been used to treat patients with melanoma. However, HDAC inhibitors are small-molecule compounds without a specific target, and their mechanism of action is unclear. Therefore, it is necessary to investigate which HDACs are required for the proliferation and metastasis of melanoma cells. Methods We used overexpression and knocking down lentivirus to clarify the influence of HDAC5 and HDAC6 in melanoma development. Also, we introduced stable HDAC5 or HDAC6 knockdown cells into null mice and found that the knockdown cells were unable to form solid tumors. Finally, we tested HDAC5 and HDAC6 expression and sub-location in clinical melanoma tissues and tumor adjacent tissues. Results In this study, and found that HDAC5 and HDAC6 were highly expressed in melanoma cells but exhibited low expression levels in normal skin cells. Furthermore, we knocked down HDAC5 or HDAC6 in A375 cells and demonstrated that both HDAC5 and HDAC6 contributed to the proliferation and metastasis of melanoma cells. Conclusions This study demonstrated both HDAC5 and HDAC6 were required for melanoma cell proliferation and metastasis through different signaling pathways. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0753-0) contains supplementary material, which is available to authorized users.

Additional file 4: Figure S4. The effect of HDAC5 and HDAC6 on metastasis didn’t depend on BRAF V600 mutation. a knocking down HDAC5 or HDAC6 in multiple BRAF V600 mutation cells. b Transwell assay to detect metastasis of A375 cells. Cell transwell assays were performed using FalconTM Cell Culture Inserts (BD353097) according to the manufacturer’s instructions. After 24–48 h of incubation, the remaining cells in the upper chamber were removed by cotton swabs. The cells on the lower surface of the membrane were fixed in 4 % paraformaldehyde and stained with 0.5 % crystal violet. Cells in at least 3 random microscopic fields (magnification ×10) were counted and photographed. All experiments were performed in duplicate and repeated 3 times.

Wound healing is an intricate process whereby the skin, or similar tissues, repairs itself after injury. Impaired wound healing with loss of temporospatial orchestration of processes remains challenging and can be highly debilitating to patients. Recently, the novel strategy of administering mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissues and other sources, has achieved high therapeutic efficiency in healing wounds. Adipose-derived stem cells (ASCs) are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors to initiate tissue regeneration. However, the exact molecular mechanism by which ASCs exert their function is not well characterized. MicroRNAs (MiRNAs) are important regulators of diverse cell functions and the cytokine network is necessary for endothelial cell migration, capillary formation, matrix metalloproteinase production, collagen synthesis and degradation. This review will focus on the significant role of MicroRNA within ASCs and how these are interlinked to the pathogenesis of wound healing.

Several clinical research studies have demonstrated that chronic cutaneous wounds can be treated with hair follicle grafts. However, the clinical outcomes of hair follicle grafting compared to split-thickness skin grafting have not been examined. This study sought to compare the clinical outcomes of patients with chronic wounds following hair follicle therapy and split-thickness skin graft therapy in a relatively large cohort of patients. Forty patients were enrolled in the study, a retrospective analysis of all patients underwent therapy with hair follicles (cohort A) and split-thickness skin grafts (cohort B) was performed. Safety, healing duration, skin quality (recipient site), scar formation (donor site) and overall postoperative outcome were analyzed. The wound sites were examined using photography at weeks 2, 8, and 12 after surgery. Five non-biased reviewers estimated the above-mentioned clinical outcomes using a five-point Likert scale. The ages and wound areas were similar between cohorts A (n=20) and B (n=20). Total wound closure was observed and adverse events were rare and controllable in both cohorts. The skin and scar quality were rated significantly higher in the hair follicle cohort than the split-thickness skin graft cohort (4.40 vs 3.45, P

Background and objectives: This preliminary laboratory-based study looks at the paracrine release from human skin cells subject to sublethal Q-switched Nd:YAG 532 nm laser irradiation. Study design/Materials and methods: Human dermal fibroblast and keratinocyte cultures were exposed to sublethal energy using the Nd:YAG 532 nm laser. Altered gene expression was then screened using RT-PCR for a range of paracrine factors known to affect melanogenesis, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), stem cell factor (SCF), melanocyte stimulating hormone (MSH), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and protease-activated receptor-2 (PAR-2). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein production. Conditioned medium was used to assess altered melanogenesis in a melanoma cell line. Results: Fibroblasts exposed to sublethal radiation showed upregulation of b-FGF, HGF and SCF. This contrasts with keratinocytes which showed upregulation of IL-6. Elevated protein levels of b-FGF and SCF were confirmed by ELISA assay. Conditioned fibroblast medium was shown to stimulate melanogenesis in a melanoma cell line. Conclusions: This preliminary laboratory study reports, for the first time, specific gene upregulation using the Q-switched Nd:YAG 532 nm laser.

We have previously shown alpha-melanocyte stimulating hormone to protect melanocytes and melanoma cells from the proinflammatory actions of tumor necrosis factor-alpha. The aim of the study was to extend this work to look into the influence of tumor necrosis factor-alpha on melanoma cell attachment, invasion, and integrin expression and ask to what extent alpha-melanocyte stimulating hormone might protect cells from tumor necrosis factor-alpha stimulation of increased integrin expression. HBL human melanoma cells were studied under resting and stressed conditions using tumor necrosis factor-alpha as a proinflammatory cytokine. Functional information on the actions of tumor necrosis factor-alpha on melanoma cells was obtained by examining the strength of attachment of melanoma cells to substrates and the ability of melanoma cells to invade through fibronectin. alpha3, alpha4, and beta1 integrin expression was detected by Western immunoblotting and the ability of alpha-melanocyte stimulating hormone to oppose the actions of tumor necrosis factor-alpha was studied on HBL cell attachment, invasion, and integrin subunit expression. Our results show that tumor necrosis factor-alpha increases the number of melanoma cells attaching to collagen (types I and IV) and tissue culture polystyrene, increases ability to invade through fibronectin, and upregulates the expression of alpha3 (28%), alpha4 (90%), and beta1 (65%) integrin subunit expression. In contrast, alpha-melanocyte stimulating hormone reduced cell attachment, invasion, and integrin expression and opposed the stimulatory effects of tumor necrosis factor-alpha. In conclusion this study provides further evidence of alpha-melanocyte stimulating hormone acting to "protect" melanoma cells from proinflammatory cytokine action. Our data support a hypothesis that an inflammatory environment would promote melanoma invasion and that the anti-invasive actions of alpha-melanocyte stimulating hormone are consistent with its working in an anti-inflammatory capacity.