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134 In vitro biological activities of a cream designed for sensitive skin treatment

  • International Hair Research Foundation
  • Giuliani, Milan, Italy
Epidermal autophagy and nucleophagy
O Akinduro
, CA Harwood
, R O’Shaughnessy
and D Bergamaschi
1 Centre for Cell
Biology and Cutaneous Research, Blizard Institute, Barts and The London SMD, QMUL,
London, United Kingdom and 2 Livingstone Skin Research Centre for Children, Institute of
Child Health, UCL, London, United Kingdom
Epidermal keratinocytes migrate through the epidermis to the granular layer where, upon
terminal differentiation they progressively lose organelles and convert into anucleate cells or
corneocytes. Autophagy is a conserved lysosomal degradation pathway which eliminates
protein aggregates and damaged organelles in order to maintain cytoplasmic homeostasis. In
keratinocytes autophagy has been described as a mechanism of senescent cell death, a stress
response leading to induction of differentiation, and a pro-survival mechanism protecting
from UV-induced damage. However, the role of autophagy in epidermis is poorly understood.
We provide a comprehensive profile of autophagy marker expression in developing
epidermis. We reinforce the importance of mTORC1 in the regulation of autophagy which is
constitutively active in epidermal granular layer where, by electron microscopy, we identified
double membrane-autophagosomes. Moreover we demonstrate that differentiating kerati-
nocytes undergo a selective form of nucleophagy characterized by accumulation of LC3/
LAMP2/p62 positive autolysosomes. By immunofluorescence we provide evidence that these
perinuclear vesicles display positivity for histone interacting protein HP1
and localize in
proximity with lamin A and B1 accumulation. We also confirmed these finding in epidermal
tissues, including newborn mice and adult human skin, where we report LC3 puncta coin-
cident with misshapen nuclei within the granular layer. This process of nuclear degradation
relies on autophagy integrity as confirmed by lack of nucleophagy upon depletion of WIPI1 or
ULK1. Finally, we report deregulated expression or location of most of the autophagic
markers and lack of LC3 in parakeratotic psoriatic skin, suggesting that impaired autophagy
contributes to the pathogenesis of psoriasis. Our findings provide the first evidence for the
existence of epidermal nucleophagy and may ultimately improve treatment options for
patients with epidermal barrier defects.
Identification of novel pathways linked to the pathogenesis of Recessive X-Linked Ichthyosis
F McGeoghan
, M Menon
, P Dewan
, M Caley
, M Donaldson
and EA O’Toole
1 Centre
for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and
Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, London, United Kingdom
Recessive X-Linked Ichthyosis (RXLI) results from a deficiency of steroid sulfatase (STS) and is
characterized by an accumulation of cholesterol sulfate in the upper layers of the epidermis,
resulting in a scaling phenotype and barrier dysfunction. To further understand the patho-
mechanisms of RXLI, we have generated an RNA-Seq data set of the transcriptome of kera-
tinocytes with siRNA-induced loss of STS. To validate these data, we generated a 3D XLRI
model using 2 telomerase-immortalized keratinocyte cell lines with stable knockdown of STS
using different lentiviral shRNA clones. Knockdown of STS was confirmed by western blotting
(P <0.001). We found the 3D model representative of RXLI in patients with acanthosis of the
epidermis on Haematoxylin and Eosin staining and altered Nile Red staining. We also saw an
increase in expression of the late differentiation marker, involucrin, in this model, which has
been observed in patient samples. Transglutaminase 1 expression and activity were also
decreased. RNA-Seq analysis showed significant dysregulation of genes involved in differ-
entiation (ACER1) and lipid metabolism (CYP4F22, UGCG). Other altered genes correlated
with extracutaneous features of RXLI, such as ALDH1A1 (corneal opacity) and OXTR (pro-
longed labour, ADHD). We validated our RNA-Seq data, using our model of RXLI and patient
samples, showing significant decreases of these genes via Western blotting and immuno-
staining. We also profiled the lipid content of our RXLI model providing novel data on lipid
changes in RXLI compared to control. In summary, we have a 3D organotypic model which
simulates the expected phenotype in vitro and this was used to further explore the molecular
pathogenesis of RXLI, providing novel pathways which can be linked to the clinical features
of the disease and may provide therapeutic targets.
Understanding the role of ABCA12 in the pathogenesis of Harlequin Ichthyosis
F Enjalbert
, P Dewan
, M Caley
, B Fell
, M Donaldson
, DP Kelsell
and EA O’Toole
1 Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medi-
cine and Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, Uxbridge, United
ATP-binding cassette transporter A12 (ABCA12), a lipid transporter, is known to be critical for
skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal
Recessive Congenital Ichthyosis (ARCI): Harlequin Ichthyosis (HI). HI patients have marked
hyperkeratosis at birth with fissuring, leading to life-threatening complications due to
increased risk of infection, trans-epidermal water and heat loss. To understand the patho-
mechanisms of HI, we used siRNA knockdown of ABCA12 in primary keratinocytes with
subsequent calcium-induced differentiation to model HI as well as a HPV- immortalised HI
patient-derived cell line. RNA-sequencing was performed on the siABCA12 primary kerati-
nocytes and siC controls. 136 genes were significantly down-regulated and 87 genes were
significantly up-regulated (FDR <0.01). Functional annotation clustering analysis performed
using DAVID showed changes in the following biological processes: inflammatory response,
endomembrane system, cytoplasmic bound vesicle/secretary granule, cell cycle, apoptosis
and epithelial development. Early expression of differentiation markers was observed in the
3D organotypic model recapitulating the HI epidermis phenotype. Further experiments were
performed to validate changes seen in nitric oxide pathway signaling genes. We found that
Arginase-1, a regulator of nitric oxide synthase activity, was down regulated in the HI
organotypic epidermis. Also, the master regulator of apoptosis and inflammation, STAT1 and
STAT1-Tyr701, were strongly upregulated in the HI patient cell line compared to control.
STAT1 activation is required for the production of nitric oxide from L-arginine. These data
suggest that nitric oxide signaling may be dysregulated in HI skin.
In vitro biological activities of a cream designed for sensitive skin treatment
D Pinto, A Benedusi, F Rinaldi and B Marzani Giuliani S.p.A., Milano, Italy
Sensitive skin syndrome is a condition of subjective and self-reported cutaneous hyper-
reactivity to environmental stimuli. This condition is characterized by a decline in epidermal
barrier function linked to an imbalance in stratum corneum intracellular lipids, while signs
such as flushing, blushing erythema are due to sub-chronic inflammation, vascular reactivity
and a hypersensitive reaction. TRPV1 was identified to contribute to this skin condition,
facilitating neurogenic inflammation leading to skin sensitivity resulting in pain or itch with a
burning sensation. TRPV1 up-regulation due to TNF-alpha stimulation highlights the corre-
lation between inflammation process and hypersensitivity skin reactions. Due to multifac-
torial pathogenesis of this skin disorders, a protective cream should be formulated to include
different actives able to improve the variety of aspects of sensitive skin. Lichtena Med
(Giuliani, Milan, Italy) contains moisturizers (betaine, inositol, trehalose, glycerin), emollients
(allantoin and Butyrospermum parkii butter), antioxidant (tocotrienols, Rosmarinus officinalis,
Olea europea extract, Calendula officinalis extracts, bisabolol, beta-sitosterol) and anti-in-
flammatory compounds (sorbityl furfural palmitate, glicyrrhetinic acid). To evaluate Lichtena
preclinical activity, we performed an in vitro test with a topical application of cream (2
) on Phenion
Full-Thickness Skin Model treated with SDS as inflammatory stimulus.
Inflammation-related genes (TNF-alpha; IL-1beta; IL-8; PTGE2) and TRPV1 gene were eval-
uated by qRT-PCR, while the DCFH-DA antioxidant assay were used for the epidermal and
dermal evaluations. The results show that Lichtena Med
has a statistically significant anti-
inflammatory activity by a down-regulation of TNF-alpha, IL-1beta, IL-8 and PTGE2 genes
associated with a significant antioxidant activity in counteracting ROS production during
inflammation process both in epidermidis and derma layers. This in vitro study suggest the
usefulness of Lichtena Med
cream in the treatment of signs associated with sensitive skin
Holographic microscopy as a new tool for imaging the effect of linear and cyclic siloxanes
(silicones) after dermal application
D Krenczkowska
, K Szymkowska
, K Mojsiewicz- Pienkowska
and E Stachowska
1 Department of Physical Chemistry, Medical University of Gdansk, Gdansk, Poland and
2 Department of Metrology and Measurement System, Poznan University of Technology,
Poznan, Poland
Siloxanes, due to beneficial properties, are common ingredients in cosmetic and pharma-
ceutical formulations. Particularly volatile methyl siloxanes are usually used as excipients.
They should give desired properties of formulation, but not destructively interact with
structures of the skin. Generally, siloxanes are regarded as biologically inert compounds.
However, after analyzing chemical structure and physicochemical properties (e.g. MW<500
Da, logP<5) it can be considered they can penetrate stratum corneum(SC). The aim of the
study was to assess the possibility of low molecular weight siloxanes: linear octamethyl-
trisiloxane L3 and cyclic decamethylocyclopentasiloxane D5 to penetrate SC and observation
of possible interaction with SC structures. In studies of human skin digital transmission ho-
lographic microscope was used for the first time. Based on comparison differences between
images of test (siloxane application) and control samples (water application) were observed.
In control samples the structure of SC is uniform. Whereas, images of test samples presented
that in the SC structure there are dark areas. It indicates a disorder of SC. Siloxanes due to
lipophilic properties penetrate by transepidermal transport through the lipid matrix causing
lipid extraction. This provides to collapse of corneocytes and formation of specific cavity,
visible as dark area. 3D images and quantitative data from measurements provide information
about the magnitude of this phenomenon. In control samples regularly corneocytes with
average size of width 20
m, and thickness of about 1
m were observed. However, in test
samples it was not possible to observe clearly distinguishable structures, possible to measure.
It has been found that the use of siloxanes influence on the human skin barrier loss. Holo-
graphic microscopy is useful method in the evaluation of SC alteration after low molecular
siloxanes application.
MiR-10a controls the proliferation and inflammatory responses of human primary
keratinocytes. MiR-10a controls the proliferation and inflammatory responses of human
primary keratinocytes
T Runnel
, E Urgard
A Rebane
1 University of Tartu, Tartu, Estoniaand 2 University of Zu
¨rich, Zu
¨rich, Switzerland
MiR-10 family genes are located in the Hox gene clusters and have been shown to be
involved in the regulation of several processes, such as cell proliferation, migration and in-
flammatory response modulation. Abnormal miR-10a/b expression has been linked to
increased invasiveness of various cancers. In immune system, miR-10a helps to control the
stability and plasticity of helper T cells. The aim of the study was to investigate how miR-10a
affects keratinocytes (KCs) and regulates their immune responses. The methods used include
miRNA profiling of KCs from atopic dermatitis (AD) patients, overexpression of miR-10a to
study changes in KC gene expression and cell cycle, KC differentiation, and luciferase assays
to verify direct targets of miR-10a. Our results show miR-10a to be upregulated in cultured
human primary KCs as well as skin biopsies from AD patients compared to healthy in-
dividuals. In human KCs, miR-10a expression was higher in proliferating conditions and
decreased in differentiation-inducing growth conditions, namely during in vitro reconstruc-
tion of the skin and high calcium environment. Overexpression of miR-10a in KCs down-
regulated the previously characterized miR-10a direct target MAP3K7 (TAK1) from the NF-
signalling pathway and hyaluronan synthase 3 (HAS3), a putative novel miR-10a direct target,
which is known to be dysregulated in AD. Subsequent luciferase assays confirmed HAS3 as a
novel direct target of miR-10a. In addition, miR-10a overexpression down-regulated many
genes linked to cell cycle regulation, epithelial development or genes in the NF-
B pathway.
Accordingly, cell cycle analysis of miR-10a-transfected KCs revealed less cells to be in the S-
phase compared to controls. We conclude that miR-10a downregulates the proliferation of
KCs through targeting multiple factors involved in the regulation of the cell cycle and has a
regulatory role for immune responses in both healthy and diseased skin.
Epidermal Structure and Function | ABSTRACTS S183
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