Gene silencing in a human organotypic skin model

Mildner, Michael; Ballaun, Claudia; Stichenwirth, Martin; Bauer, Reinhard; Gmeiner, Ramona; Buchberger, Maria; Mlitz, Veronika; Tschachler, Erwin

doi:10.1016/j.bbrc.2006.07.035

Cited by 76 publications

(90 citation statements)

References 28 publications

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“…30 However, there have been no studies to date investigating the applicability of RNAi to silence a target gene in a keloid OC model, to evaluate its efficiency as a potential antikeloid treatment in situ. Here, we demonstrate that satisfactory knockdown efficiency of PAI-1 can be achieved even in keloid OC, suggesting that other keloid-promoting genes may also be silenced in situ and no doubt encourages further investigation into the effect of target gene knockdown both for obtaining better mechanistic insights into KD pathobiology in situ and for exploring novel candidate antikeloid therapies.…”

Section: Discussionmentioning

confidence: 99%

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

Syed

Bagabir

Paus

et al. 2013

Laboratory Investigation

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Keloid disease (KD) is a common fibroproliferative disorder of unknown etiopathogenesis. Its unique occurrence in human skin and lack of animal models pose challenges for KD research. The lack of a suitable model in KD and overreliance on cell culture has hampered the progress in developing new treatments. Therefore, we evaluated the effect of two promising candidate antifibrotic therapies: ( À )-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) silencing in a long-term human keloid organ culture (OC). Four millimeters of air-liquid interface (ALI) keloid explants on collagen gel matrix in serum-free medium (n ¼ 8 cases) were treated with different modalities (EGCG treatment; PAI-1 knockdown by short interfering RNA (siRNA) and application of dexamethasone (DEX) as control). Normal skin (n ¼ 6) was used as control (only for D0 keloid-untreated comparison). Besides routine histology and quantitative (immuno-) histomorphometry, the key phenotypic and growth parameters of KD were assessed. Results demonstrated that EGCG reduced keloid volume significantly (40% by week 4), increased apoptosis (Z40% from weeks 1 to 4), and decreased proliferation (r17% in week 2). EGCG induced epidermal shrinkage, reduced collagen-I and -III at mRNA and protein levels, depleted 98% of keloid-associated mast cells, and reduced the percentage of both cellularity and blood vessel count by week 4. Knockdown of PAI-1 significantly reduced keloid volume by 28% in week 4, respectively, and reduced collagen-I and -III at both mRNA and protein levels. As expected, DEX increased keloid apoptosis, decreased keloid proliferation, and collagen synthesis, but induced connective tissue growth factor overexpression. In conclusion, using keloid OC model, we provide the first functional evidence for testing candidate antifibrotic compounds in KD. We show that EGCG and PAI-1 silencing effectively inhibits growth and induces shrinkage of human keloid tissue in situ. Therefore, the application of EGCG, PAI-1 silencing, and other emerging compounds tested using this model may provide effective treatment and potentially aid in the prevention of recurrence of KD following surgery. Keloid disease (KD) is a benign hyperproliferative dermal disease of unknown etiopathogenesis with ill-defined treatment, 1 which is unique to humans. 2 KD can arise following an abnormal wound healing process in genetically susceptible individuals. 2 The hallmark of KD is excessive deposition of extracellular matrix (ECM) in the dermis. 3 The lack of an animal model that can truly mimic human KD in vivo condition imposes limitations when investigating functional activities of potential therapeutic agents in treating KD. 4,5 However, these models have failed to provide the essential components of skin tissue that occur in vivo, such as mature ECM, blood vessels, inflammatory cells, and intrinsic and extrinsic biochemical interactions. Therefore, the ultimate aim of this study was to determine the applicability of recent research findings in existing i...

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Section: Discussionmentioning

confidence: 99%

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

Syed

Bagabir

Paus

et al. 2013

Laboratory Investigation

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“…52 Stealth small interfering RNAs (siRNAs) specific for FLG and a scrambled control were obtained from Invitrogen. 53 siRNA transfection and preparation of organotypic skin cultures were performed as described previously.…”

Section: Organotypic Culturesmentioning

confidence: 99%

“…53 siRNA transfection and preparation of organotypic skin cultures were performed as described previously. 52 Briefly, thirdpassage keratinocytes were transfected using Lipofectamine 2000 (Invitrogen). Five milliliters of OPTI-MEM medium (Gibco/Invitrogen) was mixed with 50-L Lipofectamine 2000 and 130 L of a 20 mol/L siRNA solution.…”

Section: Organotypic Culturesmentioning

confidence: 99%

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

Gruber

Elias

Crumrine

et al. 2011

The American Journal of Pathology

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219

238

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Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix.These results clarify how FLG mutations impair epidermal permeability barrier function.

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“…Gene-specific siRNAs were also used to additionally knockdown KLK5 or KLK7. These KC were used together with primary human dermal fibroblasts to establish full skin equivalents (SE) based on a published protocol (9).…”

Section: Experimental Designmentioning

confidence: 99%

SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7

Wang

Olt

Schoefmann

et al. 2014

Experimental Dermatology

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Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lympho-epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast-populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin-like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin-like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.Abbreviations: 3D, 3-dimensional; DSC1, desmocollin 1; DSG1, desmoglein 1; FLG, filaggrin; H&E, haematoxylin-eosin; KC, keratinocytes; kd, knockdown; KLK, kallikreins; LEKTI, lympho-epithelial Kazal type inhibitor 5; NS, Netherton syndrome; sc, stratum corneum; SCCE, stratum corneum chymotrypsin-like enzyme; SCTE, stratum corneum trypsin-like enzyme; SE, skin equivalents; siRNA, small interfering ribonucleic acid; SPINK5, serine protease inhibitor of kazal type 5.

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Gene silencing in a human organotypic skin model

Cited by 76 publications

References 28 publications

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7

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