Apoptosis In Human Dermal Fibroblasts Research Articles

Photobiomodulation and photodynamic therapy-induced switching of autophagy and apoptosis in human dermal fibroblasts.

Nowadays, photobiomodulation (PBM) in combination with chemotherapy or other therapeutic approaches is an attractive adjuvant modality for cancer treatment. Targeted destruction of cancer cells is one of the main advantages of photodynamic therapy (PDT). We have shown in previous studies that the combination of PBM at 808nm and hypericin-mediated PDT increases PDT efficacy in human glioblastoma cells U87 MG. The study presented here shows significant differences between U87 MG and non-cancerous human dermal fibroblasts (HDF) cells treated by PBM and PDT. This study focuses on mitochondria because PBM mainly affects these organelles. We demonstrated that an interplay between mitochondrial and autophagic proteins plays a crucial role in the response of HDF cells to PBM and PDT. Fluorescence microscopy, flow cytometry, and Western blot analysis were used to examine the autophagic profile of HDF cells after these treatments. An increase in ubiquitin, SQSTM1, LC3BII, and cytochrome c was accompanied by a decrease in M6PR, ATG16L1, and Opa1 in HDF cells exposed to PBM and PDT. Overall, we observed that the switching of autophagy and apoptosis is dose-dependent and also occurs independently of PBM in HDF cells after hypericin-mediated PDT. However, PBM might preferentially induce autophagy in noncancer cells, which might escape apoptosis under certain conditions.

Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation

Although UVB radiation is mainly absorbed by the epidermis, ~5–10% of its photons reach and affect the upper part of the dermis. Physiologically relevant UVB doses, able to provoke erythema, induce apoptosis in human dermal fibroblasts in vitro, as well as in the dermis of SKH-1 mice. Given the sparse and even contradictory existing information on the effect of UVB radiation on dermal fibroblasts’ viability, aim of this work was to unravel the crucial signaling pathways regulating the survival of UVB-treated human dermal fibroblasts. We found that UVB radiation immediately stimulates the phosphorylation of MAPK family members, as well as Akt, and is genotoxic leading to the delayed ATM-p53 axis activation. Akt phosphorylation after UVB radiation is EGFR-mediated and EGFR inhibition leads to a further decrease of viability, while the Akt activator SC79 rescues fibroblasts to an extent by a mechanism involving Nrf2 activation. The known Nrf2 activator sulforaphane also exerts a partial protective effect, although by acting in a distinct mechanism from SC79. On the other hand, inhibition of JNKs or of the ATM-p53 axis leads to a complete loss of viability after UVB irradiation. Interestingly, JNKs activation is necessary for p53 phosphorylation, while the ATM-p53 pathway is required for the long-term activation of JNKs and Akt, reassuring the protection from UVB. Although UVB radiation results in intense and prolonged increase of intracellular ROS levels, classical anti-oxidants, such as Trolox, are unable to affect Akt, JNKs, or p53 phosphorylation and to reverse the loss of fibroblasts’ viability. Collectively, here we provide evidence that the main viability-regulating UVB-triggered biochemical pathways act synergistically towards the protection of human dermal fibroblasts, with EGFR/Akt and Nrf2 serving as auxiliary anti-apoptotic machineries, while JNKs/ATM-p53 activation and interplay being overriding and indispensable for the perpetuation of cellular defense and the maintenance of cell viability.

Protective Effects and Molecular Mechanism of Total Flavonoids from Lycium Barbarum Leaves on Photoaged Human Dermal Fibroblasts.

Objective To investigate the effects and corresponding mechanisms of total flavonoids (TFL) from Lycium barbarum leaves on photoaged human dermal fibroblasts (HDFs). Methods Crude TFL was extracted with 70% ethanol, and a Rutin standard curve was drawn using the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry method to calculate its yield and mass concentration. After that, the photoaging HDFs model was established by UVA combined with 8-MOP. CCK-8 was performed to assess the influence of TFL on the proliferation of HDFs and photoaging HDFs. β-galactosidase (SA-β-gal) staining and activity assays were performed to evaluate the activity of SA-β-gal and the rate of SA-β-gal-positive cells in HDFs cells. The level of skin ECM proteins and oxidative stress-related substances in HDFs cells of each group was determined by ELISA and biochemical detection, respectively. Apoptosis of HDFs in each group was assessed by flow cytometry. The expressions of MAPK signaling pathway-related proteins in HDFs were detected by western blot. Results The yield rate of TFL extracted by 70% ethanol was 41.9%, and its purity rate was 34.6%. TFL at 25, 50, and 100 μg/mL was able to greatly promote the proliferation of HDFs. A photoaged HDFs model was successfully constructed by combining UVA irradiation at 9 J/cm2 and 8-MOP at 50 mg/L. TFL treatment could significantly inhibit apoptosis, SA-β-gal-positive cell staining rate, SA-β-gal activity, lactate dehydrogenase (LDH) leakage, and malondialdehyde (MDA) content in photoaged HDFs. Further, TFL increased the proliferative activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, type I collagen (Col I), hydroxyproline (HYP), and hyaluronic acid (HA) level of photoaged HDFs in a dose-dependent manner. Additional experiments suggested that TFL played a protective role by downregulating MAPK signaling pathway activity in photoaged HDFs cells. Conclusion TFL could inhibit oxidative stress and apoptosis, promote cell proliferation and the level of ECM-related component proteins, and participate in antiphotoaging in a concentration-dependent manner. The protective role of TFL in photoaged HDFs might be related to its inhibition of MAPK signaling pathways.

Chlorogenic Acid Prevents UVA-Induced Skin Photoaging through Regulating Collagen Metabolism and Apoptosis in Human Dermal Fibroblasts.

Skin aging is categorized as chronological aging and photo-aging that affected by intrinsic and extrinsic factors. The present study aimed to investigate the anti-aging ability and its underlying mechanism of chlorogenic acid (CGA) on human dermal fibroblasts (HDFs). In this study, CGA specifically up-regulated collagen I (Col1) mRNA and protein expressions and increased the collagen secretion in the supernatant of HDFs without affecting the cell viability, the latter was also demonstrated in BioMAP HDF3CGF system. Under ultraviolet A (UVA)-induced photoaging, CGA regulated collagen metabolism by increasing Col1 expression and decreasing matrix metalloproteinase 1 (MMP1) and MMP3 levels in UVA-irradiated HDFs. The activation of transforming growth factor-β (TGF-β)-mediated Smad2/3 molecules, which is crucial in Col1 synthesis, was suppressed by UVA irradiation and but enhanced at the presence of CGA. In addition, CGA reduced the accumulation of UVA-induced reactive oxygen species (ROS), attenuated the DNA damage and promoted cell repair, resulting in reducing the apoptosis of UVA-irradiated HDFs. In conclusion, our study, for the first time, demonstrate that CGA has protective effects during skin photoaging, especially triggered by UVA-irradiation, and provide rationales for further investigation of CGA being used to prevent or treat skin aging.

Role of haematopoietic cell-specific protein 1-associated protein X-1 gene in lipopolysaccharide-induced apoptosis of human dermal fibroblasts.

Wound healing may be disrupted by lipopolysaccharide (LPS)-induced mitochondrial dysfunction, inflammation, and excessive oxidative stress, which can lead to undesirable consequences. The haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) is a mitochondrial matrix protein that regulates mitochondrial function. This study aimed to comprehensively identify the role of HAX-1 in the inhibition of LPS-induced mitochondrial dysfunction and apoptosis in human dermal fibroblasts (HDFs). HAX-1 expression was assessed in the HDF-a cell line using real-time polymerase chain reaction, western blotting, and immunohistochemical staining. The viability, migration, and apoptosis of HDF-a cells were evaluated using the water-soluble tetrazolium-1 assay, transwell assay, and flow cytometry analysis, respectively. Mitochondrial function was evaluated based on reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm). Our results demonstrated that LPS stimulation markedly repressed HAX-1 expression in HDFs and silencing of HAX-1 led to mitochondrial ROS accumulation, ΔΨm disruption, and abnormal mitochondrial morphology. Accordingly, overexpression of HAX-1 or administration of metformin enhanced mitochondrial fusion and normalized mitochondrial dynamics, thereby reversing LPS-induced mitochondrial dysfunction, fibroblast apoptosis, and viability and migration inhibition in HDF-a cells. These data support a mechanism wherein HAX-1 plays a crucial role in LPS-induced fibroblast apoptosis in a mitochondria-dependent manner.

Advanced Glycation End Products (AGEs) Induce Apoptosis of Fibroblasts by Activation of NLRP3 Inflammasome via Reactive Oxygen Species (ROS) Signaling Pathway.

BackgroundType 2 diabetes impairs the healing process and induces apoptosis of fibroblasts, which are thought to be involved in this process. We investigated the possible mechanisms involved in AGEs-induced apoptosis of human dermal fibroblasts.Material/MethodsWe examined the expression of apoptosis-related proteins in fibroblasts isolated from human diabetic wounds. Human dermal fibroblasts exposed to AGEs were used to study the links among apoptosis, ROS, and NLRP3 inflammasome activation. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. Cleaved caspase-8, cleaved caspase-3, BAX, Bcl-2, and NLRP3 inflammasome expression were measured by Western blot analysis. ROS generation, cell viability, and cell apoptosis were assessed.ResultsWe observed a higher level of cleaved caspase-8 and cleaved caspase-3 expression in fibroblasts isolated from human diabetic wounds compared with controls. AGEs decreased the proliferation of cells in a concentration-dependent and time-dependent manner. The exposure of fibroblasts to AGEs significantly increased the number of cells in early and late apoptosis stages. AGES-induced human dermal fibroblasts showed high expressions of cleaved caspase3, cleaved caspase8, and Bax. Treatment with AGEs induced the expression of NLRP3, caspase-1, and ASC. AGES-induced apoptosis was blocked by BAY 11–7082, an inhibitor of the NLRP3 inflammasome. AGEs increased the production of ROS in fibroblasts, and its apoptogenic effect was blocked by NAC.ConclusionsAGEs cause apoptosis of fibroblasts by inducing the generation of ROS and activating the NLRP3 inflammasome. In vivo experiments are needed to confirm these results.

X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis

Sex dimorphism in cell response to stress has previously been investigated by different research groups. This dimorphism could be at least in part accounted for by sex-biased expression of regulatory elements such as microRNAs (miRs). In order to spot previously unknown miR expression differences we took advantage of prior knowledge on specialized databases to identify X chromosome-encoded miRs potentially escaping X chromosome inactivation (XCI). MiR-548am-5p emerged as potentially XCI escaper and was experimentally verified to be significantly up-regulated in human XX primary dermal fibroblasts (DFs) compared to XY ones. Accordingly, miR-548am-5p target mRNAs, e.g. the transcript for Bax, was differently modulated in XX and XY DFs. Functional analyses indicated that XY DFs were more prone to mitochondria-mediated apoptosis than XX ones. Experimentally induced overexpression of miR548am-5p in XY cells by lentivirus vector transduction decreased apoptosis susceptibility, whereas its down-regulation in XX cells enhanced apoptosis susceptibility. These data indicate that this approach could be used to identify previously unreported sex-biased differences in miR expression and that a miR identified with this approach, miR548am-5p, can account for sex-dependent differences observed in the susceptibility to mitochondrial apoptosis of human DFs.

Comparing the effect of gold and silver nanoparticles on human dermal fibroblasts using Cellomics high-content screening system

Event Abstract Back to Event Comparing the effect of gold and silver nanoparticles on human dermal fibroblasts using Cellomics high-content screening system Yan Huang1* and Xiaoying Lü1* 1 Southeast University, State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, China Introduction: Gold nanoparticles (GNPs) and silver nanoparticles (SNPs) are widely used in the field of biomedicine, and their biosafety must not be overlooked. Cellomics high-content screening (HCS) system is a combination of cell biology with automated high resolution microscopy and robotic handling, providing detailed analyses of cytotoxicity of biomaterials based on parallel analyses of multiple markers. The aim of this study is to compare the influence of GNPs and SNPs on cell membrane permeability, cell adhesion, mitochondrial membrane potentials, and apoptosis of human dermal fibroblasts (HDFs) with the HCS system. Materials and methods: GNPs and SNPs were synthesized by water-phase reaction and their size and morphology were observed by transmission electron microscope (TEM). HDFs were exposed to 200μM GNPs and SNPs for 1, 4, 8, 24, 48 and 72h, respectively, and their effects were analyzed by the HCS system. Cells were dyed with Sytox and membrane permeability was assayed by calculating relative fluorescent intensity of Sytox. For focal adhesion observation, HDFs were stained using a FAK100 kit. Mitochondrial membrane potentials were monitored by determining relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates by JC-1 probing. Cell apoptosis detection were carried out using PI/Hoechst dyeing method. Results and discussion: TEM images showed that GNPs and SNPs were generally spherical with size of ~20 nm. HCS analyses showed that relative fluorescent intensity of Sytox in GNP-treated HDFs had no obvious difference compared to untreated cells; while significiant increase was detected after cells were treated with SNPs for 48 and 72h, indicating that SNPs destroyed cell membranes after 48h. Fig. 1 displays the focal adhesion of untreated, GNP- or SNP-treated HDFs for 72h. No distinct alteration in number, area of focal adhesion and nucleus were observed in GNP-treated cells; while in SNP-treated groups, the amount and area of focal adhesion was significantly reduced and was accompanied with a decrease of number and area of nucleus, indicating that SNPs inhibited cell adhesion and proliferation. JC-1 staining results revealed that mitochondrial membrane potentials in HDFs did not changed after GNPs treatment, while the potentials increased after treatment of SNPs for 8h, implying that SNPs could induce early apoptosis. PI/Hoechst dyeing results suggested that SNPs induced HDFs later apoptosis after 4h, while GNPs did not. In summary, GNPs did not induce toxicity at the cellular level, while SNPs destroyed membrane, restrained cell adhesion, increased mitochondrial membrane potentials, induced apoptosis, inhibited cell proliferation, and finally led to cytotoxicity. Fig. 1. Focal adhesion of HDFs observed on HCS platform (a) Untreated HDFs, (b) HDFs exposed to GNPs for 72h, (c) HDFs exposed to SNPs for 72h. Green: Focal adhesion, Blue: nucleus. Conclusion: With the Cellomics HCS system, the influence of GNPs and SNPs on HDFs was compared, and the toxicity effect of SNPs was illustrated. The obtained results had a good consistency with our previous conclusions from multi-biomics data analysis[1],[2]. National Natural Science Foundation of China(31170910, 31271012); Natural Science Foundation of Jiangsu Province of China(BK20150599); 973 project (2009CB930000); The Specialized Research Fund for the Doctoral Program of Higher Education of China (20100092110027)

ZnO nanoparticles induce apoptosis in human dermal fibroblasts via p53 and p38 pathways

The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continues to expand. Zinc oxide nanoparticles (ZnO NPs) are used in various applications, including catalysis, electronics, biosensors, medicine, paints, sunscreens and cosmetics, thus it is important to understand the biological effects and risks of ZnO NPs. This study was designed to investigate the apoptosis induction by ZnO NPs via mitogen-activated protein kinase p38 and cell cycle checkpoint protein p53 pathways in human dermal fibroblasts. MTT-based cell viability assay showed a significant decrease in cell survivorship after ZnO NP exposure, and phase contrast images revealed that ZnO NP treated cells had lower density and a rounded morphology. Apoptosis induction was confirmed by the annexin V assay and Western blot analysis showed the up-regulation of p53 and phospho-p38 proteins. Furthermore, in ZnO NP exposed cells, p53 protein was phosphorylated at Ser33 and Ser46 sites known to be phosphorylated by p38. Our results suggest that ZnO NPs have the potential to induce apoptosis in human dermal fibroblasts via p53–p38 pathways.

Effects of Extracellular Matrix Glycosylation on Proliferation and Apoptosis of Human Dermal Fibroblasts Via the Receptor for Advanced Glycosylated End Products

The balance between proliferation and apoptosis of skin cells is responsible for skin turnover and the success of the wound healing process. Recent reports have shown that advanced glycosylation end product (AGE) formation participates in dermatologic problems in diabetes. However, the effect on proliferation and apoptosis of dermal fibroblasts remains unclear. The aim of this study was to investigate the effects of dermal microenvironment glycosylation on the balance of cellular proliferation and apoptosis. Histology and immunohistochemical staining were performed on type II diabetic and nondiabetic skin tissue specimens to determine the distributions of proliferating cell nuclear antigen, apoptotic cells, AGEs, and receptors for AGEs (RAGEs). Matrix secreted by cultured human fibroblasts was glycosylated by 0.5 M D-ribose. RAGE-blocking antibodies were applied to inhibit the interaction of RAGE and AGEs in this system and then cell viability, cell cycle phase distribution, and apoptosis were measured. Diabetic skin has degenerative, loosely arranged collagen and increased apoptotic cells compared with normal skin. Expression of AGE and RAGE in diabetic skin tissue increased. Glycosylated matrix induced cell cycle arrest and apoptosis of cultured dermal fibroblasts, whereas application of RAGE-blocking antibodies redressed these changes. The accumulation of glycosylated extracellular matrix in diabetic skin tissue is a critical mediator of cellular function. Mediation of RAGE affects the balance of cellular proliferation and apoptosis, which confirms that diabetic wounds possess atypical origin in the repair process.

Fas-Mediated Apoptotic Signal Transduction in Keloid and Hypertrophic Scar

The pathogenesis of keloid and hypertrophic scar is less well understood because of the lack of animal models. The cell-surface Fas receptor, which is widely distributed in skin components, has been shown to be an important factor that induces apoptosis in human dermal fibroblasts. In this report, the authors performed a comparative study on apoptotic signal transduction in fibroblasts derived from keloids and hypertrophic scars. Fibroblasts harvested from 10 patients with keloids and hypertrophic scars were used for this study. At first, apoptosis induction using Fas antibody and C2-ceramide were evaluated using electron microscopy and flow cytometry. Second, the expression of the apoptosis-related proteins Fas and Bcl-2 were measured by means of flow cytometry. Third, ceramide was measured by quantitative derivatization to ceramide-1-phosphate using diacylglycerol kinase and [gamma-32P] ATP. Moreover, intercellular Ca2+ was investigated using confocal microscopic analyses. In contrast to hypertrophic scar-derived fibroblasts, those derived from keloids are significantly resistant to Fas-mediated apoptosis. The intercellular ceramide and Ca were not activated. There were no significant differences in the level of expression of Bcl-2 between the two groups, but Fas expression was higher in keloid than in hypertrophic scar. Fibroblasts from the two groups were susceptible to ceramide-induced apoptosis. Blocking of the Fas-mediated apoptotic pathway in keloids occurs upstream of the second messenger. The abnormal Fas-induced apoptosis in keloids may account for the imbalance of proliferation and apoptosis. Enhancement of Fas sensitivity could be a promising therapeutic target.

Hexavalent chromium disrupts the actin cytoskeleton and induces mitochondria-dependent apoptosis in human dermal fibroblasts

Exposure to hexavalent chromium causes various adverse effects including deep skin ulcerations and allergic dermatitis. Because of many potential intracellular targets for hexavalent chromium toxicity, its mechanisms of action are not entirely understood. To investigate the role of the cytoskeleton and mitochondria in this process, primary human dermal fibroblasts were exposed to various concentrations of potassium chromate for 24 h. The followed markers included cell motility, cytoskeletal organization, oxidative stress, mitochondrial activity and activation of the apoptotic cascade. Potassium chromate (1.5–45 μM) induced time- and concentration-dependent cell shrinkage, reorganization of cytoskeleton and loss of motile activity in fibroblasts. In some cells this was followed by membrane blebbing. Dynamic changes in cell morphology were accompanied with the loss of mitochondrial membrane potential, increased oxidative stress and release of cytochrome c. Apoptosis was confirmed by detection of activated caspase-3 and nuclear fragmentation. The results indicate that in fibroblasts hexavalent chromium-induced damage to cytoskeleton and mitochondria might occur concurrently at relatively early stages of exposure. Furthermore, alterations of these targets seem to activate mitochondria-dependent and- independent apoptosis.

Retinol, at concentrations greater than the physiological limit, induces oxidative stress and apoptosis in human dermal fibroblasts.

We have investigated the dose (in the range of microM) and time-dependent effects of four different retinoids (retinol, retinal, retinoic acid and retinol palmitate) on human dermal fibroblasts cultivated in vitro. Retinol and retinal, at a concentration of 20 microM, caused cell damage as evaluated by lactate dehydrogenase activity released into the culture medium. The oxidised glutathione (GSSG)/reduced glutathione (GSH) ratio and malondialdehyde production indicated that 20 microM of retinol provoked oxidative stress in the cultivated human fibroblasts. In the first 8 h after retinol treatment the levels of p53 and Bax proteins as well as caspase 3 activity increased, suggesting apoptotic cell death during the first hours of treatment. If the retinol treatment exceeded 18-24 h we observed necrotic cell death. Vitamin E and coenzyme Q(10) had a protective effect against oxidative stress generated by retinol. Both antioxidant molecules reduced retinol uptake, and in the case of vitamin E the expression of CRABP-II mRNA was induced, providing a plausible explanation for its protective effect.