915 Impaired cutaneous wound healing in tumor necrosis factor stimulated gene-6 deficient mice

Full text

915
Impaired cutaneous wound healing in tumor necrosis factor stimulated gene-6
deficient mice
S Shakya
1
, J Mack
2
and EV Maytin
3
1 Cleveland Clinic, Cleveland State University, Cleve-
land, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical Engi-
neering, Cleveland Clinic, Cleveland, OH
Non-healing, chronic wounds, such as diabetic ulcers, are a health major burden worldwide.
Cutaneous wound healing is a complex process that involves a multitude of events occurring
concurrently, such as inflammation, epithelial and fibroblast proliferation and migration,
angiogenesis, and extracellular matrix (ECM) deposition and remodeling. Hyaluronan (HA) is
a key ECM glycosaminoglycan molecule known to regulate wound healing by mediating
inflammation, cell proliferation and migration, angiogenesis and fibrosis. Tumor necrosis
factor stimulated gene-6 (TSG-6) protein is an enzyme that modifies HA by transferring heavy
chains (HC) from Inter-
a
-Trypsin Inhibitor (I
a
I) to form complexes of HC and HA (HC.HA),
which are considered to stabilize the ECM and are believed to be more adherent to in-
flammatory cells than HA itself. Our preliminary analysis of wild type (WT) mouse skin
showed that TSG-6 and HC.HA is constitutively present in normal skin, and increased in
wounds. Little is known about the roles of HC.HA or TSG-6 in cutaneous wound healing. To
study this, we made full-thickness excisional wounds in mice lacking TSG-6 (TSG-6KO mice)
and analyzed HC.HA, wound closure and inflammation. We found that compared to WT,
HC.HA complexes are absent in TSG-6 KO unwounded and wounded skin indicating there is
no redundancy or compensation for lack of TSG-6. Wound closure is significantly delayed in
TSG-6 KO mice at days 3, 5 and 7 post-wounding. Finally, neutrophil recruitment is differ-
entially regulated in TSG-6 KO wounds; while neutrophil recruitment was delayed in early
wounds, an exacerbated response was seen at later time points. We propose that the delayed
neutrophil recruitment in early wounds is due to the absence of HC.HA in the luminal gly-
cocalyx of blood vessels in TSG-6 KO mice. To investigate this further, we will use an in-vitro
culture model using freshly isolated neutrophils and an endothelial cell line.
916
Epidermal regeneration processes are enhanced by carob extract in vitro
MJ Flagler, M Hare, J Henry and R Osborne Procter & Gamble, Mason, OH
The appearance of aging skin can be improved via cosmetic procedures such as chemical
peels and laser treatments which damage the skin surface. Similar to cosmetic procedures, we
hypothesize appearance benefits can be obtained from the application of materials which
activate elements of the wound healing response (cell migration and proliferation). Two in
vitro test methods were leveraged to evaluate a botanical extract (Carob) for the potential to
trigger a wound healing response in vitro: (1) Electrical Cell-substrate Impedance Sensing
(ECIS; Applied Biophysics, Troy, NY); and (2) Incucyte ZOOM Scratch Wound Assay (Essen
Bioscience, Ann Arbor, MI). The ECIS assay utilizes thermal wounding (electrical current) to
quantitatively evaluate in vitro cell migration/proliferation enhancement, and cell adhesion
(barrier) properties. By contrast, the Incucyte platform uses physical wounding (scratch
wound) to quantitatively evaluate in vitro cell migration/proliferation enhancement in
response to cellular damage. In the ECIS platform, Carob extract enhanced the rate of in vitro
wound healing in hTERT keratinocytes while also increasing cell-to-cell adhesion in a dose-
dependent manner. Carob extract also significantly increased the rate of in vitro scratch
wound closure in the IncuCyte scratch wound assay in hTERT keratinocytes. These results
suggest Carob extract is able to significantly improve in vitro skin regeneration via activation
of a wound healing response.
917
CD44 and p38 participate in regulating the pro-fibrotic capability of dermal
fibroblasts during skin wound healing
Y Wang
1
, J Mack
2
and EV Maytin
3
1 Cleveland Clinic Lerner Research Institute, Shaker
Heights, OH, 2 Cleveland Clinic, Cleveland, OH and 3 Dermatology and Biomedical En-
gineering, Cleveland Clinic, Cleveland, OH
During wound healing, fibroblasts synthesize extracellular matrix molecules, e.g. collagen
and hyaluronan (HA). CD44 is the major cell surface receptor for HA on fibroblasts. Our
previous work showed that wound closure is accelerated in double-knockout mice that lack
the HA-synthetic enzymes Has1 and Has3, but retain a functional Has2. Morphological
analysis (Masson trichrome stains) of post-wounding skin revealed that dermal maturation
occurs sooner in Has1/3 null mice than WT mice. To seek mechanistic insights for these
observations, skin fibroblasts from Has1/3 null or WT mice were isolated and cultured in
vitro. Compared to WT cells, the Has1/3 null fibroblasts had higher Has2 gene expression,
larger pericellular HA coat, upregulated TGF-
b
activity, and more abundant collagen I and
a
-
smooth muscle actin (
a
-SMA). CD44 gene expression was also increased. However, neither
the removal of extracellular HA by hyaluronidase, nor knockdown of Has2 gene expression
by RNA interference (RNAi) had any impact upon
a
-SMA gene expression in Has1/3 fibro-
blasts. On the other hand, CD44 RNAi raised intrinsic TGF-
b
activity and enhanced TGF-
b
-
induced
a
-SMA gene expression in WT and Has1/3 fibroblasts. The ability of fibroblasts to
contract collagen gel was impaired by CD44 RNAi, and cell morphology was altered
(changes in focal adhesion size and actin stress fiber arrangement). Chemical inhibition of the
activity of p38 MAPK effectively diminished the phenotypic difference in
a
-SMA protein
abundance between WT and Has1/3 null cells, but failed to abrogate induction of
a
-SMA by
CD44 RNAi. Together, these data suggest that the fibrotic capacity of fibroblasts is enhanced
in Has1/3 null mice in an HA-independent but p38-dependent manner. CD44 plays a
complex role in regulating
a
-SMA gene expression, actin stress fiber reorganization, and
cellular contractility in dermal fibroblasts.
918
Tissue engineered human hair follicles from genetically, environmentally and
extrinsically reprogrammed dermal papilla cells
A Coffman
1
, H Abaci
1
, JC Chen
2
, E Wang
1
, Y Doucet
2
, Z Guo
2
and AM Christiano
2
1 Columbia University Medical Center, New York, NY and 2 Columbia University,
New York, NY
Human skin equivalents (HSEs) have provided an effective therapy for patients with significant
skin loss, however they still have limitations including poor viability and lack of skin ap-
pendages. We recently improved the viability of skin grafts by establishing a method to
micropattern vasculature in HSEs. However, it still remains a prevailing challenge to engineer
functional HSEs with hair follicles; since cultured human dermal papilla cells (DPs) in vitro
lose their hair-inducing capacity. We have previously shown that the molecular memory of
human DPs can be partially restored when DPs are grown as 3D spheroids. Here, we aimed
to expand our previous work and achieve complete restoration of DP phenotype and func-
tionality by leveraging microenvironmental, genetic, and extrinsic approaches. We devel-
oped an innovative tissue engineering strategy and used 3D-printing technology to create hair
follicle-like microchannels on HSEs. These microchannels allowed DPs to spontaneously
form 3D spheroids and generate an optimal conformation of cells to initiate epidermal-
mesenchymal interactions. To promote the hair inductivity of the DPs, we overexpressed Lef-
1, which we previously identified as a master regulator of the inductive DP gene signature.
Incorporating Lef-1 transfected DPs in HSEs significantly promoted hair follicle differentia-
tion. Prolonged culture of these constructs yielded well-defined hair follicle layers, subse-
quently resulting in the growth of hair fibers. We further enhanced hair follicle induction in
HSEs testing various exogenous small molecules targeting Wnt - and Jak-STAT signaling. This
ability to regenerate an entire hair follicle from cultured human cells will have an over-
whelming impact on the medical management of patients with significant skin loss.
919
Wnt3a-conditioned media of human bone marrow-derived mesenchymal stem
cells induces human dermal fibroblast wnt3a gene expression
J McBride, L Rodriguez-Menocal, A Candanedo and E Badiavas University of Miami,
Department of Dermatology and Interdisciplinary Stem Cell Institute, Miami, FL
Wnts are a family of secreted, lipidated glycoproteins that regulate stem cell self-renewal and
differentiation during embryonic development. Wnt ligands are implicated in modulation of
skin development and homeostasis during adulthood. Recently, both canonical and “alter-
native” Wnt pathways have been implicated in processes that both promote and antagonize
skin wound repair. Here, we report new evidence that Wnt3a, produced by a donor cell type
(bone marrow-derived mesenchymal stem cells), may positively upregulate its own expres-
sion in another cell type (dermal fibroblasts). Treatment of primary human dermal fibroblasts
with serum-free Wnt3a-conditioned media of primary human bone marrow-derived mesen-
chymal stem cells (BM-MSC Wnt3a CM) significantly induced human Wnt3a gene expression
in the dermal fibroblasts, compared to control BM-MSC CM. This BM-MSC Wnt3a CM
stimulates canonical Wnt/T-cell factor/Lymphoid enhancer factor/beta-catenin signaling and
stimulates fibroblast proliferation. This model suggests a novel feed-forward mechanism for
canonical Wnt signaling between BM-MSCs and dermal fibroblasts. Canonical Wnt signals
may promote fibroblast proliferation during the proliferative phase of wound healing, but also
promote fibrotic scarring during the remodeling phase of wound healing. This feed-forward
amplification signal should be balanced by negative canonical Wnt regulators from numerous
autocrine and paracrine sources to reduce wound fibrosis during wound remodeling. This
result highlights the complexity of paracrine Wnt signaling regulation between the bone
marrow and dermis.
920
CCR6-deficient mice have delayed skin wound closure and reduction in
peri-wound macrophage infiltration
L Anderson
1
,XWu
2
, S Simon
3
and S Hwang
4
1 UC Davis, Sacramento, CA, 2 UCDavis,
Sacramento, CA, 3 UC Davis, Davis, CA and 4 UC Davis School of Medicine, Sacramento,
CA
Psoriasis is a chronic inflammatory disease afflicting up to 3% of Caucasians worldwide. A
murine model of psoriasis shows that CCR6
+
gd
T cells are recruited to psoriatic skin in part
by CCL20 and that these cells produce proinflammatory cytokines, IL-17 and IL-20, which
promotes disease pathogenesis. Notably, CCR6
-/-
mice fail to develop psoriasis in response to
induction by exogenous IL-23, raising interest in CCR6 as a relevant biological target for this
disease. Because the CCR6/CCL20 pathway has been shown to regulate corneal wound
repair, we sought to determine if CCR6-deficient mice had defects in epithelial repair in the
skin. Utilizing a 6mm full thickness skin wound on the dorsum of CCR6
-/-
or congenic WT
mice, we investigated the importance of CCR6/CCL20 signaling in wound healing. Specif-
ically, we examined changes in wound size over time and determined changes in the
recruitment of CCR6
+
gd
T cells and macrophages as wounds healed. A reduction in rate of
wound closure was observed in CCR6
-/-
compared to WT mice with the greatest difference
observed at day 6, where CCR6
-/-
wounds were 80% of initial wound size whereas WT
wounds were 20% of initial wound size (P-/- mice compared to WT at days 1, 3 and 5 post-
wounding. In addition, a 2-fold increase in the number of circulating
gd
T cells was detected
in CCR6
-/-
mice compared to WT mice at baseline (p-/- wounds correlates with less macro-
phage invasion. While all wounds in CCR6-/- mice did eventually heal, these preliminary
studies reveal a potential side effect of CCL20/CCR6 inhibition as a therapy for psoriasis and
thus may be important in the design of future clinical trials. Additional studies are underway
to elucidate the signaling mechanisms that link
gd
T cells to recruitment of macrophages to
wounds.
ABSTRACTS | Tissue Regeneration and Wound Healing
S158 Journal of Investigative Dermatology (2017), Volume 137