Skin Dermal Fibroblasts Research Articles

Increase in cyclooxygenase-2 (COX-2) expression in keratinocytes and dermal fibroblasts in photoaged skin.

Interleukins and NFκ-B are involved in the development of inflammatory reactions. It has been suggested that these proteins are important contributing factors in the process of photoaging of skin. Moreover, interleukins and NFκ-B are known to be capable of inducing expression of cyclooxygenase 2 (COX-2). Expression of COX-2 in various populations of skin cells has not been examined in the specific processes of aging. The study aimed at evaluating COX-2 expression in skin samples originating from patients with chronologically aged and photoaged skin at various stages of skin aging. Immunohistochemical analysis of COX-2 reactivity was conducted on samples originating from 52 women undergoing surgery for reasons other than skin pathology. Our study demonstrated that COX-2 expression in keratinocytes and fibroblasts was significantly higher in skin samples affected by photoaging than in samples affected by endogenous aging or obtained from younger individuals. The results indicate that COX-2 may be involved in the pathogenesis of the photoaging process. Inhibition of expression or activity of the enzyme may find application in photoaging treatment and/or prophylaxis.

NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-β1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-β1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-β1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-β1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-β1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-β1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.

제주 구멍갈파래 가수분해물에 의한 노화된 섬유아세포 증식 및 콜라겐 합성증진 효과

피부 섬유아세포는 인간 피부의 주요 콜라겐 생산 세포이다. 노화가 진행되면, 섬유아세포에서의 콜라겐 생산이 감소되고, matrix metalloproteinase-1 (MMP-1)에 의해 시작되는 콜라겐 조각화가 증가된다. 즉 섬유아세포의 콜라겐 항상성의 불균형으로 인해 피부 collagenous, 세포외기질(ECM)의 구조와 기능이 변형되어, 피부노화가 촉진되는 것이다. Cysteine rich protein 61 (CCN1)는 CCN family의 일부이며, 인간피부의 섬유아세포에서 콜라겐 항상성을 조절하는 단백질이다. 노화된 인간 피부 섬유아세포에서의 CCN1 과 발현은 실질적으로 유형 I procollagen 생성을 감소시킴과 동시에 MMP-1의 발현을 증가시켜 섬유의 콜라겐 저하를 일으킨다. 그리고 노화된 섬유아세포는 노화 전 섬유아세포에 비해 증식률이 감소한다. 본 연구에서 만들어 사용한 복제 노화 피부 섬유아세포는 유형 I procollagen의 생성량이 감소하였고, MMP-1의 발현 수준이 증가하는 특징을 나타냈다. 또한 CCN1 단백질의 발현이 증가되고, 증식률이 감소하는 특징을 나타냈다. 가수분해 구멍갈파래 추출물은 노화 전 섬유아세포에서 새로운 콜라겐의 합성을 촉진하고 자외선에 의해 증가된 MP-1의 발현을 감소시켜 광노화를 개선하는 물질로 알려져 있다. 본 연구에서는 이러한 활성을 나타내는 가수분해 구멍갈파래 추출물을 사용하여, 복제 노화 피부 섬유아세포에서 가수분해 구멍갈파래 추출물에 의한 CN1 단백질의 발현 억제 여부를 조사하였으며, 이들 추출물은 배양된 복제 노화 피부 섬유아세포에서 유형 I procollagen의 생성을 증가시켰으며, MMP-1 발현을 억제시키는 것을 확인하였다. 또한, 콜라겐 항상성을 조절하는 단백질인 CN1 발현을 크게 감소시켰으며, 노화세포의 증식률을 증가시켰다. 이 결과는 복제 노화 섬유아세포가 in vitro 자연 노화모델로 화장품 원료 활성 연구에 사용될 수 있음을 말한다. 그리고 가수분해 구멍갈파래 추출물은 광노화 뿐 아니라 자연노화를 개선하는 피부미용제로 주름개선 기능성 화장품에 사용가능 하다는 것을 의미한다. Skin dermal fibroblast is the major collagen-producing cell type in human skin. As aging process continues in human skin, collagen production is reduced and fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This imbalance of collagen homeostasis impairs the structure and function of dermal collagenous extracellular matrix (ECM), thereby promoting skin aging. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis in primary human skin dermal fibroblast cells. It is known in aging fibroblast cells that elevated CCN1 expression substantially reduces type I procollagen and concurrently increases MMP-1, which initiates fibrillar collagen degradation. And proliferation rate of aging fibroblast cells is reduced compared to the pre-aging fibroblast cells. In this study, we confirmed that the replicative senescence dermal fibroblast cells increased the expression levels of MMP-1 and decreased the production of type I procollagen. Our results also showed that the replicative senescence dermal fibroblast cells increased in the expression of CCN1 and decreased in the proliferation rate. Hydrolyzed Ulva pertusa extracts are the materials to improve photo-aging by reducing the expression of MMP-1 that was increased by ultraviolet and by promoting the synthesis of new collagen from fibroblast cells. In this study, we also investigated the hydrolyzed U. pertusa extract to see whether it inhibits CCN1 protein expression in the senescence fibroblasts. Results showed that the hydrolyzed U. pertusa extract inhibited the expression of MMP-1 and increased the production of type I procollagen in the aging skin fibroblast cells cultured. In addition, the proteins that regulate collagen homeostasis CCN1 expression were greatly reduced. The hydrolyzed U. pertusa extract increased the proliferation rate of the aging fibroblast cells. These results suggest that replicative senescent fibroblast cells may be used in the study of cosmetic ingredients as a model of the natural aging. In conclusion, the hydrolyzed U. pertusa extract can be used in anti-wrinkle functional cosmetic material to improve the natural aging skin care as well as photo-aging.

Radio Electric Conveyed Fields Directly Reprogram Human Dermal Skin Fibroblasts toward Cardiac, Neuronal, and Skeletal Muscle-Like Lineages

Somatic cells can be directly reprogrammed to alternative differentiated fates without first becoming stem/progenitor cells. Nevertheless, the initial need for viral-mediated gene delivery renders this strategy unsafe in humans. Here, we provide evidence that exposure of human skin fibroblasts to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields at a radiofrequency of 2.4 GHz, afforded remarkable commitment toward cardiac, neuronal, and skeletal muscle lineages. REAC induced the transcription of tissue-restricted genes, including Mef2c, Tbx5, GATA4, Nkx2.5, and prodynorphin for cardiac reprogramming, as well as myoD, and neurogenin 1 for skeletal myogenesis and neurogenesis, respectively. Conversely, REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 h, followed by a downregulation at later times. The REAC action bypassed a persistent reprogramming toward an induced pluripotent stem cell-like state and involved the transcriptional induction of the NADPH oxidase subunit Nox4. Our results show for the first time the feasibility of using a physical stimulus to afford the expression of pluripotentiality in human adult somatic cells up to the attainment of three major target lineages for regenerative medicine.

Cysteine-rich Protein 61 (CCN1) Domain-specific Stimulation of Matrix Metalloproteinase-1 Expression through αVβ3 Integrin in Human Skin Fibroblasts

Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVβ3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVβ3 integrin. The interaction of VWC domain with integrin αVβ3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVβ3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

Human Skin-Derived Fibroblasts Acquire In Vitro Anti-Tumor Potential after Priming with Paclitaxel

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p<0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs. Keywords: Drug uptake, Mesenchymal stem cells, Skin dermal fibroblasts, Stromal cells, Taxol

Human Skin-Derived Fibroblasts Acquire In Vitro Anti-Tumor Potential after Priming with Paclitaxel

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p < 0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs.

A human monoclonal antibody 264RAD targeting αvβ6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo

αvβ6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvβ6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvβ6, and inhibits binding to all ligands including the latency-associated peptide of TGF-β. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvβ8, but not the integrins α5β1, αvβ3, αvβ5 and α4β1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvβ6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-β-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-β by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvβ6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvβ6 integrin, with some activity against αvβ8 integrin, that reduces both tumour growth and metastasis.

Cysteine‐rich protein 61 (CCN1) mediates replicative senescence‐associated aberrant collagen homeostasis in human skin fibroblasts

Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

Polyester/cellulose acetate composites: Preparation, characterization and biocompatible

AbstractThe biocompatibility, morphology, and mechanical thermal properties of composite materials composed of maleic anhydride‐grafted polylactide (PLA‐g‐MA) and cellulose acetate (CA) were evaluated. Composites containing maleic anhydride‐grafted PLA (PLA‐g‐MA/CA) exhibited noticeably superior mechanical properties due to greater compatibility between the two components. The dispersion of CA in the PLA‐g‐MA matrix was highly homogeneous as a result of ester formation, and the consequent creation of branched and cross‐linked macromolecules, between the carboxyl groups of PLA‐g‐MA and hydroxyl groups in CA. The human skin dermal fibroblasts (FBs) seeded on these two novel series membranes to verify the wound dressing characterization properties. With time‐dependent course, the FBs proliferation demonstrated a better increasing performance on the series membranes of PLA/CA than PLA‐g‐MA/CA. The immunofluorescent staining illustrated FBs with normal morphological features. The collagen amount from FBs on the PLA/CA series was 25% higher than that seeded on regular culture‐plates after 7 days incubation. With CA content from 0 to 20%, the collagen amount increased apparently and up to the position of CA20%. Otherwise, with PLA‐g‐MA membranes, the collagen amount showed moderate stimulations. The SEM image presented secreted collagen from FBs on the PLA/CA membrane, indicating the bio‐functional properties of these membranes. The above result analysis, this PLA‐g‐MA/CA composites has good mechanical properties and biocompatible, in the future when use adjusts the formula according to the functionality, may increase the product utility. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Low Herpesvirus Entry Mediator (HVEM) Expression on Dermal Fibroblasts Contributes to a Th2-Dominant Microenvironment in Advanced Cutaneous T-Cell Lymphoma

LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes) is a ligand for HVEM. LIGHT-HVEM interactions are important in T helper type 1 (Th1) immune responses. In some cases with early stages of cutaneous T cell lymphoma (CTCL), IL-2, IFN-γ, and Th1 chemokines are expressed in lesional skin, while IL-4, IL-5, and Th2 chemokines are dominant in advanced CTCL. In this study, we investigated roles of LIGHT and HVEM in the microenvironment of CTCL. LIGHT enhanced production of Th1 chemokines, such as CXC chemokine ligand (CXCL) 9, CXCL10, and CXCL11, from IFN-γ-treated dermal fibroblasts via phosphorylation of inhibitor κBα. Messenger RNA levels of these chemokines were increased in lesional skin of early CTCL. Interestingly, while LIGHT expression in CTCL skin correlated with disease progression, HVEM expression was significantly decreased in advanced CTCL skin. HVEM was detected in dermal fibroblasts in early CTCL skin, but not in advanced CTCL skin in situ. These results suggest that low HVEM expression on dermal fibroblasts in advanced CTCL skin attenuates expression of Th1 chemokines, which may contribute to a shift to a Th2-dominant microenvironment as disease progresses.

Sesamol modulates ultraviolet-B-induced apoptotic and inflammatory signaling in human skin dermal fibroblasts

Aim: Sesamol (SM), a dietary phenolic phytochemical, has been shown to reduce ultraviolet-B (UVB) mediated oxidative damage. The aim of the present study was to investigate the protective mechanism of SM against UVB-induced photoaging, inflammatory and apoptotic signaling in human skin dermal fibroblasts, adult (HDFa) in vitro . Materials and Methods: In this study, we examined the effect of SM on UVB radiation-induced loss of mitochondrial membrane potential (ΔΨm), DNA fragmentation, cell cycle modulation, inflammatory markers [tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB] expression, pro-apoptotic (p53, Bax and caspase-3), and anti-apoptotic marker (Bcl-2) expression in HDFa. We also investigated the effect of SM and/or UVB radiation on matrix metalloproteinase (MMP-2 and MMP-9) activation by gelatin zymograpy in HDFa. Results and Conclusion: SM pretreatment prevented UVB-induced ΔΨm alteration, DNA fragmentation and down-regulated the expressions of apoptotic (p53, Bax and caspase-3) and inflammatory markers (TNF-α and NF-κB) in HDFa. SM also prevented the activation of MMP-2 and MMP-9 in a concentration-dependent manner. Our data indicated the ability of SM to block UVB-induced inflammatory and apoptotic signaling in HDFa. However, further detailed mechanistic approach warrants before claiming this compound for photoprotection.

Human hair follicle dermal cells and skin fibroblasts show differential activation of NF‐κB in response to pro‐inflammatory challenge

The underlying mechanism of immune privilege in hair follicle cell dermal papilla (DP) and sheath (DS) populations is not well understood, and the responsiveness of hair follicle dermal cells to pro-inflammatory challenge presently remains unknown. In this work, we describe acute NF-κB activation in human DS, DP and dermal fibroblast (DF) cells challenged with TNF-alpha and IL1-beta. In contrast, the DS and DP cells revealed an unexpected tolerance to bacterial LPS challenge relative to DF cells. Understanding follicle cell responses to typical pro-inflammatory stimuli is critical for diseases where collapse of hair follicle immune privilege is observed, and to further applications in autologous stem cell/wound healing therapeutics.

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

Targeting of Mitochondria-Endoplasmic Reticulum by Fluorescent Macrocyclic Compounds

BackgroundUseful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes.MethodologyFour polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen2N4 which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy.ConclusionOur study reveals that compounds Me2[28]py2N6, cryptphen, [16]phenN2, [30]phen2N6, have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.

Cigarette smoke-induced early growth response-1 regulates the expression of the cysteine-rich 61 in human skin dermal fibroblasts.

Tobacco smoke is known to be an element contributing to accelerate premature skin ageing. Cysteine-rich 61 (Cyr61) is a member of the connective tissue factor CCN (Cyr61, CTGF and Nov) family, and early growth response-1 (Egr-1) is a generally expressed member of the zinc-finger family of transcription factors. To investigate the regulatory potential of Egr-1 on expression of Cyr61 by smoking, this study examined the hypothesis that cigarette smoke-induced Egr-1 induces expression of Cyr61 in human skin dermal fibroblasts (HSDF). HSDF were exposed to different concentrations of cigarette smoke extract (CSE) for 24 h; a cytotoxicity assay was then performed for the detection of cell proliferation. Results of Western blot and reverse transcription-polymerase chain reaction (RT-PCR) showed that CSE induces a transient synthesis of Egr-1 in HSDF. Cyr61 mRNA and protein levels showed a marked increase in a time-dependent manner after CSE exposure. Following transfection with an Egr-1 overexpression vector, HSDF showed increased activity of the Cyr61 promoter in a dose-dependent manner. Using Egr-1 interfering RNA, we confirmed that CSE-induced Cyr61 expression was dependent on Egr-1 expression. Findings of this study indicate that Egr-1-dependent induction of Cyr61 may contribute to premature skin ageing by smoking.

Comparison of hair dermal cells and skin fibroblasts in a collagen sponge for use in wound repair

The adult hair follicle has well-defined dermal and epithelial populations that display distinct developmental properties. The follicular dermal cells, namely the dermal papilla and dermal sheath, are derived from the same mesenchymal cells as dermal fibroblasts and therefore, we believed that follicular cells could be useful sources of interfollicular keratinocytes and fibroblast for skin wound repair. In this study, we evaluated the relative effect of various mesenchymal-derived cells on wound healing following skin injury. Human dermal cells, including two different follicular dermal cells and skin fibroblasts were cultured in collagen sponges and compared with respect to wound healing. Results indicated that there was no significant difference in wound contraction and angiogenesis among the cell types. Further, dermal sheath cells exhibited relatively poor results compared with other cells in new collagen synthesis. Finally, basement membrane reformation and new collagen synthesis for the dermal papilla cell grafts was superior to those of the dermal sheath cells or fibroblasts.

CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts

Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

Development of Histocompatible Primate‐Induced Pluripotent Stem Cells for Neural Transplantation

Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.

Bone Marrow‐Derived Mesenchymal Stromal Cells Inhibit Th2‐Mediated Allergic Airways Inflammation in Mice

Bone marrow-derived mesenchymal stromal cells (BMSCs) mitigate inflammation in mouse models of acute lung injury. However, specific mechanisms of BMSC actions on CD4 T lymphocyte-mediated inflammation in vivo remain poorly understood. Limited data suggests promotion of Th2 phenotype in models of Th1-mediated diseases. However, whether this might alleviate or worsen Th2-mediated diseases such as allergic asthma is unknown. To ascertain the effects of systemic administration of BMSCs in a mouse model of Th2-mediated allergic airways inflammation, ovalbumin (OVA)-induced allergic airways inflammation was induced in wild-type C57BL/6 and BALB/c mice as well as in interferon-γ (IFNγ) receptor null mice. Effects of systemic administration during antigen sensitization of either syngeneic or allogeneic BMSC on airways hyperreactivity, lung inflammation, antigen-specific CD4 T lymphocytes, and serum immunoglobulins were assessed. Both syngeneic and allogeneic BMSCs inhibited airways hyperreactivity and lung inflammation through a mechanism partly dependent on IFNγ. However, contrary to existing data, BMSCs did not affect antigen-specific CD4 T lymphocyte proliferation but rather promoted Th1 phenotype in vivo as assessed by both OVA-specific CD4 T lymphocyte cytokine production and OVA-specific circulating immunoglobulins. BMSCs treated to prevent release of soluble mediators and a control cell population of primary dermal skin fibroblasts only partly mimicked the BMSC effects and in some cases worsened inflammation. In conclusion, BMSCs inhibit Th2-mediated allergic airways inflammation by influencing antigen-specific CD4 T lymphocyte differentiation. Promotion of a Th1 phenotype in antigen-specific CD4 T lymphocytes by BMSCs is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNγ-dependent process.