Content uploaded by Zimei Wu
Author content
All content in this area was uploaded by Zimei Wu on Mar 29, 2017
Content may be subject to copyright.
Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters
Travis Badenhorst1, Darren Svirskis1, Mervyn Merrilees2, Liane Bolke3 and Zimei Wu1*
1School of Pharmacy, Faculty of Medical and Health Sciences, New Zealand
2School of Medical Sciences, Faculty of Medical and Health Sciences, New Zealand
3Dermatest GmbH, Münster, Germany
*Corresponding author: Zimei Wu, Senior Lecturer in Pharmaceutics, School of Pharmacy, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand,
Tel: 6499231709; Fax: 64 93677192; E-mail: z.wu@auckland.ac.nz
Received date: November 16, 2016; Accepted date: December 20, 2016; Published date: December 22, 2016
Copyright: © 2016 Badenhorst T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is an endogenous tripeptide-copper complex involved
in collagen synthesis and is used topically as a skin anti-aging and wound healing agent. However, its biological
effects are yet to be fully elucidated.
Objectives: To investigate the effects of GHK-Cu on gene expression of metalloproteinases (MMPs) and tissue
inhibitors of metalloproteinases (TIMPs), and on production of collagen and elastin by human adult dermal
fibroblasts (HDFa); and to investigate the effectiveness of topical application of GHK-Cu on wrinkle parameters in
volunteers.
Methods: Cultured HDFa were incubated with GHK-Cu at 0.01, 1 and 100 nM in cell culture medium. Gene
expression (mRNA) for MMP1, MMP2, TIMP1 and TIMP2 in treated and control HDFa was measured by RT-PCR.
Cellular production of collagen and elastin was measured colourmetrically using commercial assay kits. Correlations
between gene expression and collagen and elastin production were determined. A randomised, double-blind clinical
trial involving twice daily application of GHK-Cu, encapsulated in lipid-based nano-carrier, to facial skin of female
subjects (n= 40, aged 40 to 65) was run over 8 weeks. The formulation vehicle (a serum) and a commercial
cosmetic product containing Matrixyl® 3000, a lipophilic GHK derivative, were used as controls.
Results: GHK-Cu significantly increased gene expression of MMP1 and MMP2 at the lowest concentration whilst
simultaneously increasing the expression of TIMP1 at all the tested concentrations. All examined concentrations of
GHK-Cu increased both collagen and elastin production. An increase of the mRNA expression ratio of TIMPs to
MMPs was associated with an increase in collagen/elastin production. Application of GHK-Cu in nano-carriers to
facial skin of volunteers significantly reduced wrinkle volume (31.6%; p=0.004) compared to Matrixyl® 3000, and
significantly reduced wrinkle volume (55.8%; p<0.001) and wrinkle depth (32.8%; p=0.012) compared to control
serum.
Conclusions: GHK-Cu significantly increased collagen and elastin production by HDFa cells depending on the
relative mRNA expression of their TIMP(s) over MMP. Topical application of GHK-Cu with the aid of nano-carriers
reduced wrinkle volume to a significantly greater extent than the vehicle alone or a commercial product containing
Matrixyl 3000®, a GHK lipophilic derivative.
Keywords: GHK-tripeptide; Metalloproteinases (MMP); Tissue
inhibitors of metalloproteinases (TIMP); Cell culture; Delivery; Skin
barrier
Introduction
e peptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is gaining
interest as an anti-aging and wound healing bioactive agent [1-3].
GHK is capable of up and downregulating over 4000 genes [4].
Previously, the eects of GHK-Cu on collagen production and
metalloproteinase (MMP) expression have been investigated in
cultured rat broblasts and in rat wound healing models [5-7]. GHK-
Cu functions as an activator of tissue remodelling and increases
secretion of MMP2 and a number of tissue inhibitors of
metalloproteinases (TIMP1 and TIMP2) in cultured broblasts [7,8].
MMP1, 8 and 13 degrade mainly brillar collagens whilst gelatinases
MMP2 and 9, act on type IV collagen in the basement membrane and
elastin. Increased expression of MMPs typically occurs in heightened
inammatory responses that are usually marked by opposing
inhibitory processes [9]. TIMPs tightly control MMP activities through
competitive irreversible inhibition, thereby controlling the breakdown
and re-synthesis of the extracellular matrix [10,11].
erefore, increased TIMP expression in the skin may have anti-
wrinkle benets. While this has yet to be determined, topical
application of GHK-Cu has resulted in benecial eects on wrinkles
[12]. A 12 week trial of topically administered GHK-Cu in 71
volunteers demonstrated improvements in ne lines, viscoelastic
properties, thickness and density of the skin, without irritation [12].
Other trials report signicant improvements in skin appearance [13],
Journal of Aging Science Badenhorst et al., J Aging Sci 2016, 4:3
DOI: 10.4172/2329-8847.1000166
Research Article OMICS International
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
increased dermal keratinocyte proliferation and increased pro-collagen
synthesis [14]. Maquart et al. found dose related eects of GHK-Cu,
including increases in dry weight, total protein, collagen and
glycosaminoglycan content in rat skin [15].
Topical application of GHK-Cu, amongst numerous other peptides,
is widely promoted in the cosmetic industry [16]. Our previous pre-
formulation studies, however, showed the logD of GHK-Cu at pH 4.5
and 7.4 to be -2.49 ± 0.33 and -2.49 ± 0.35, respectively, suggesting the
tripeptide is highly hydrophilic [17]. erefore, while this peptide may
have considerable biological potential, the ecient trans-epidermal
delivery of GHK-Cu is challenging based on its physicochemical
properties. Ideally, compounds should have a moderate oil-water
partition coecient (log P) of between 1-3 and few polar centres in
order to permeate into the skin [18]. To overcome the epidermal
barrier, in this present study GHK-Cu was formulated into a lipophilic
nano-carrier that improves delivery into the skin. Increased
lipophilicity may also be achieved by combining GHK with a lipophilic
moiety. For example, Matrixyl 3000®, containing a chemical
combination of GHK and palmitic acid, is currently used as an active
component in commercial cosmetic products.
ere are potential dierences associated with GHK-Cu application
to rat and human broblasts [19]. is study therefore aimed to
determine the eect of GHK-Cu, for the rst time, on human adult
dermal broblasts (HDFa), using collagen, elastin, and mRNA
expression of MMP1, MMP2, TIMP1 and TIMP2 as biological
markers. Further, the eect of GHK-Cu on wrinkle parameters was
evaluated in volunteers, comparing a product containing Matrixyl
3000®, and a serum vehicle control, in a randomised double-blind,
split-face, trial. Wrinkle depth and volume changes were used as
endpoints. Given our previous study [17] demonstrated that GHK-Cu
is highly hydrophilic, a lipid-based nano-carrier system was employed
for delivery into the skin. is trial is the rst to investigate the
biological eect of GHK-Cu formulated into a nano-carrier.
Materials and Methods
Materials
GHK-Cu was purchased from Salkat Ltd (Auckland, New Zealand).
Snowberry New Zealand Limited provided the New Radiance Face
Serum (NRFS - nano-carrier containing GHK-Cu in a serum vehicle)
and CONTROL (the serum vehicle without GHK-Cu or nano-carrier).
Strivectin SD Advanced Intensive Concentrate (SSID) was purchased
online. All Taqman gene expression assay kits, reagents, 18S
housekeeper genes, SuperScript complimentary DNA Synthesis kit and
PureLink RNA mini kit were purchased from Applied Biosystems (Life
technologies, Auckland, New Zealand). Reagents for agarose
preparation (SeaKem LE agarose) were purchased from Lonza
(Auckland, New Zealand). Human adult primary dermal broblasts
(HDFa) were purchased from Invitrogen (Invitrogen, USA). All
phosphate buered saline (PBS, 10 mM) was freshly prepared (8 g
NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1 litre water and
adjusted to pH 7.4 with HCl. All water used was prepared by reverse
osmosis.
Cell culture of primary human dermal broblasts
Fibroblasts, passage numbers between 4 and 8, were seeded in 6-
well growth plates at a density of 30,000 cells per well. Cells were
incubated in 3 mL DMEM with 1.5% FBS and 50 U ml-1 each of
penicillin and streptomycin for 24 h at 37°C in a Heracell 150i
incubator (ermo Fisher Scientic, Victoria, Australia) maintaining a
5% CO2 atmosphere with >80% relative humidity. e cells were
incubated with GHK-Cu solutions in DMEM for 24 hours. e nal
concentrations of GHK-Cu were 0.01, 1 and 100 nM, a range
previously reported to produce a response in MMP and TIMP
expression in
in vitro
rat broblasts [7]. Control cultures received an
additional volume of water (without the GHK-Cu) at the same time as
the treated cultures. Each condition was examined in triplicate, with
each culture tested three times. Following removal of medium, cells
were processed for RNA extraction.
RNA extraction and PCR
RNA extraction was performed using a RNA extraction kit based on
the manufacturer’s protocol (Biocolor, Carrickfergus, Ireland). Briey,
control and treated cells were washed with PBS three times, cells
transferred to RNase-free tubes and centrifuged at 2000 x g for 5
minutes to obtain a cell pellet. e supernatant was removed and 0.6
mL lysis buer added along with 1% 2-mercaptoethanol to re-suspend
the pellet. e dispersion was mixed with a 70% ethanol solution at 1:1
(v/v) and centrifuged in a spin cartridge tube and attached collection
tube. e collection tube was washed twice with buer to purify the
RNA, which was eluted aer a nal wash with RNase-free water.
RNA quality and quantity assessment
Concentration and integrity of extracted RNA was quantied using
a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, USA).
Purity criteria was >1.8 for 260/280 ratio (the
ratio
of absorbance at
260 nm and 280 nm) and >2 for the 260/230 ratio (Nanodrop technical
support documentation). Conrmation of quality was by Agarose gel
electrophoresis. To prepare the gel substrate, 2.6 g of SeaKem LE
agarose was dissolved in 65 ml tris-borate-EDTA (TBE) buer at 80°C,
placed onto the frame, and allowed to set for a minimum of 30
minutes. TBE buer was added, covering the gel completely, before
PCR product (10 μL) was added to the wells along with 5 μL Blue Juice
Loading Buer. A 100V potential dierence was placed across the gel
and le for 60 minutes. e gel was stained with 100 ml TBE buer
containing 5 μL of a 10 mg mL-1 ethidium bromide (EtBr) solution.
Aer 15 minutes the EtBr was washed o with water and imaged using
a GelDoc EZ Imager (Biorad Laboratories, Auckland, New Zealand).
Reverse transcription (cDNA synthesis) and RT-qPCR
e extracted RNA was reverse transcribed using the SuperScript
complimentary DNA (cDNA) Synthesis kit using the manufacturer’s
protocol (Applied Biosystems). Briey, the reaction mixture comprised
14 μL of RNA free water, 4 μL of 5× VILOTM master mix and 2 μL of
10× SuperScript® Enzyme Mix. Negative reverse transcription (RT)
samples were produced as a control. e samples were incubated in an
Applied Biosystems Gene Amp PCR 9700 for 10 min at 25°C, 120 min
at 42°C and 5 min at 85°C, sequentially.
To quantify cDNA, real time quantitative polymerase chain reaction
(RT-qPCR) was used [20,21]. RT-qPCR was performed with a
predesigned mix (RTMIX) consisting of 5 μL Master Mix, 0.5 μL Gene
Expression Assay, 0.5 μL 18S (a housekeeper gene), and 2 μL of RNase-
free water.
e RTMIX (8 µl) was combined with 2 μL of cDNA sample and
placed into a 384-well MicroAmp® plate. Each cDNA sample was
prepared in triplicate and each condition was measured 3 times in 3
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 2 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
separate culture samples. Aer loading all the samples, the MicroAmp®
plate was sealed and centrifuged for 1 minute at 2000 rpm. e plate
was placed into a Sequence Detection System (Life technologies,
Auckland, New Zealand) for DNA amplication. 6-Carboxyuorescein
(FAM) was used as the uorophore for each Taqman gene expression
probe whilst 4,7,2′-trichloro-7′-phenyl-6-carboxyuorescein (VIC)
was used for the 18S housekeeper gene [22]. Amplication consisted of
four-cycle stages: 50°C for 2 minutes, 60°C for 30 seconds, 95°C for 10
minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 30
seconds. To calculate the relative gene expression the results from RT-
qPCR were normalized using the 2-∆∆Ct method [23].
Collagen and elastin quantication
Cells were seeded in 12-well plates (surface area 3.77 cm2 per well)
at a density of 1 × 105 cells per well. Each well contained 3 mL cell
culture medium without bovine serum. Aer the initial 24 h to allow
cell settling and attachment, each well was supplemented with GHK-
Cu solution (in water, 100 µL). Cells were treated for 48 and 96 h.
Control samples were supplemented with water. Following treatment,
cell culture medium was collected for collagen measurement, while the
cells were detached by incubation with 250 µL of trypsin for 10
minutes at 37°C for elastin measurement.
Collagen measurement: Total soluble collagen content in culture
medium was measured using the Sircol soluble collagen assay kit as
described by manufacturer’s protocol (Biocolor, Carrickfergus, Ireland)
[24]. To isolate the collagen, 1 mL of cell culture medium was placed
into a 1.5 mL Eppendorf tube and mixed with a 200 µL aliquot of the
provided isolation and concentration reagent (polyethylene glycol
TRIS-HCl buer, pH 7.6). Following vortex mixing for 30 seconds,
samples were incubated at 4°C overnight to allow collagen to
precipitate. Samples were then treated as per manufacturer’s
instructions (Biocolor, Carrickfergus, Ireland) and transferred to a 96
well plate for absorbance measurement at 555 nm. Reagent blanks
were also measured for absorbance. e concentration of collagen was
then calculated using a standard curve.
Elastin measurement: e cell suspension was transferred to a
microcentrifuge tube and pelleted at 12,000 × g. 1.0 M oxalic acid
solution (100 µL) was added to the pellet making a nal concentration
of 0.25 M oxalic acid solution and heated to 100°C for 1 h to convert
insoluble elastin to soluble α-elastin. e samples were then treated as
per manufacturer’s instructions (Biocolor, Carrickfergus, Ireland) and
transferred to a 96 well plate for reading at 513 nm [24]. Reagent
blanks (250 µL) including oxalic acid, PBS and water served as
controls. Absorbance at 513 nm from these control reagents was
subtracted from the nal readings of the sample, providing a reading
for elastin. Absorbance values were converted into concentrations
using a standard curve.
Eects on human facial wrinkle parameters
e trial was conducted at Dermatest GMBH, a cosmetic research
institute, with 40 female volunteers (aged from 40 to 65 years) over the
course of 8 weeks. All test products were supplied as identically
packaged coded containers with investigators and subjects blinded to
treatments. Prior to application all the subjects underwent a
dermatological examination and a 10-day period of no cosmetic use.
All participants gave informed consent.
Participants were randomly placed into two treatment groups:
Group 1) NRFS serum, the Nano-carrier enhanced GHK-Cu serum
and a product containing Matrixyl® 3000 (SSID) as a positive control,
and Group 2) NRFS serum and CONTROL, the latter contained no
GHK-Cu or nano-carrier, acting as a negative control. All subjects
applied the NRFS serum twice each day (in the morning and again in
the evening) to the right side of the face, and either the SSID (Group 1)
or CONTROL (Group 2) to the le side of the face according to the
same instructions. e participants were instructed to not apply other
skin care products to the test areas during the course of the trial.
Measurement of wrinkle depth and volume
e Phaseshi Rapid
in vivo
Measurement of the Skin system
(PRIMOS, GF Messtechnik GmbH, Teltow, Germany) was used as a
three-dimensional analytical instrument for this study. e principle of
this instrument has been previously described by Jaspers et al. [25].
Briey, a digital stripe projection technique is used as an optical
measurement process. A parallel stripe pattern is projected onto the
skin over the wrinkle area and depicted on the CCD chip of a digital
camera connected to an evaluation computer. e measurement head
is then moved close to the immobilized head of the participant
(Caneld Scientic Inc., NJ, U.S.A). e parallel projections are then
distorted by the elevation dierences on the skin and a three-
dimensional eect recorded. e distortions provide a qualitative and
quantitative measurement of the skin prole. ey are digitised and
quantitatively evaluated using soware attached to the PRIMOS
system. e measurement area was a 30 × 40 mm region, located as
close as possible to the corner of the eye. A single large wrinkle was
identied on each subject and measured for wrinkle depth and volume
at 4 (NRFS and SSID) and 8 weeks (NRFS, SSID and CONTROL).
Measurements of the same wrinkle at 4 and 8 weeks aer treatment
were compared with initial measurements.
Data analysis of
in vivo
trial data
R package lme (R version 3.0.2) was used to calculate statistical
signicance using regression analysis. e signicance level was set at
0.05. For the primary analysis, comparing the NRFS serum and SSID, a
multiple linear mixed model was used with random intercepts for each
patient, to take account of the grouping by participants in the data. In
the multiple linear mixed model, the groups being compared (NRFS vs
SSID and NRFS vs CONTROL), with the right side of subject’s face
belonging to one group and the le side of the same subject’s face
belonging to the other treatment were made exactly comparable with
regard to starting wrinkle depth, and eliminated the individual
biological variation of the subjects. e equivalent analysis was
performed for wrinkle volume.
Percentages reported in this study were calculated by averaging the
percentage changes for each individual in the trial. e individual
changes were calculated from the relative change percentages; e.g.,
starting skinfold depth (833.4 µm) was compared against the 8-week
depth measurement (722.1 µm). e dierence (111.3 µm) was
expressed as a 13.35% change.
Results
RNA quality and quantity
All samples for RNA analysis met the purity criteria conrmed by
Agarose gel electrophoresis. e 260/280 ratio was 1.95 ± 0.15 and the
260/230 ratio was 2.12 ± 0.08, over all the samples examined.
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 3 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
Eects of GHK-Cu on mRNA expression of MMPs and
TIMPs
Aer 24 hours, expression of MMP1, MMP2, TIMP1 and TIMP2 in
the GHK-Cu treated cell lines, relative to untreated controls, all
showed a concentration dependency eect (Figure 1) with increased
expression at lower GHK-Cu concentrations except for the TIMP2. For
MMP1 and MMP2, interestingly, only the lowest concentration (0.01
nM) treatment resulted in signicantly increased expression (p=0.03).
TIMP1 expression was signicantly increased at all concentrations,
with a concentration eect, while TIMP2 expression was signicantly
decreased at higher concentrations (1 and 100 nM).
Figure 1: Eect of GHK-Cu on gene expression of MMP1, MMP2, TIMP1 and TIMP2 in HDFa cultures aer incubation for 24 hours. Data
are means ± SD, n=3 from three individual experiments. * Denotes p<0.05 statistical dierence from control (untreated cells).
Eects of GHK-Cu on collagen and elastin production
Both collagen (0.0726x+0.05, R2=0.983) and α-elastin (0.0178x +
0.05, R2=0.984) standard curves showed a linear relationship between
the absorbance and concentration within the ranges tested (5-15 µg/ml
and 12.5-50 µg/ml respectively).
Production Concentration
of GHK-Cu
(nM)
Treatment (hours)
0 48 96
Collagen 0 (control) 6.97 ± 1.0 7.55 ± 0.3 15.29 ± 0.4
0.01 8.64 ± 0.3 18.04 ± 1.8*
1 8.14 ± 0.7 17.07 ± 1.4*
100 8.53 ± 0.5 16.63 ± 1.6*
α-Elastin 0 (control) 36.57 ± 3.8 79.03 ± 1.5 200.4 ± 2.5
0.01 82.84 ± 4.0 257.97 ± 2.5**
1 80.48 ± 5.6 271.09 ± 4.3**
100 84.08 ± 2.4 268.2 ± 2.6**
Table 1: Collagen and elastin levels (µg/ml) of HDFa cells treated with
GHK-Cu. Data are means ± SD, n=3. * p<0.05 and ** p<0.017
compared to the control.
Aer treated with GHK-Cu solutions, the production of collagen or
α-elastin by broblasts only slightly increased at 48 hours compared
with the non-treated cells (Table 1). For both collagen and α-elastin,
GHK-Cu signicantly increased secretion over the controls at 96
hours. ere was an inverse dose dependent response for collagen
production at 96 hours. Alpha-elastin was increased by approximately
30% at all concentrations but without a clear concentration
dependency.
As the expression of MMP1 and TIMP1 reduced, with increasing
concentration of GHK-Cu, there was a corresponding decrease in
collagen. A similar trend did not occur for α-elastin.
Eect of GHK-Cu on wrinkle parameters
e Snowberry New Radiance Face Serum (NRFS), SSID (Strivectin
SD Advanced Concentrate), and CONTROL (Control vehicle) were
well tolerated by 39 of the 40 subjects throughout the eight-week
application period. One participant experienced minor unwanted skin
reactions on both the right and le side of their face aer application
of the NRFS serum and SSID. is subject ceased application and
symptoms resolved without medical treatment. Wrinkle depth and
volume changes at four and eight-weeks are summarised in Table 2.
Comparisons and statistical signicance between treatment groups are
given in Table 3.
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 4 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
Treatment Wrinkle
parameter
(weeks)
Percent change
from baseline
NRFS serum Depth (4) -18.3 ± 10.3
(n=19) Depth (8) -26.8 ± 12.8
Volume (4) -17.2 ± 8.1
Group 1 Volume (8) -25.8 ± 9.4
SSID Depth (4) -15.9 ± 8.6
(n=19) Depth (8) -22.4 ± 8.5
Volume (4) -16.8 ± 8.6
Volume (8) -20.0 ± 7.8
NRFS serum Depth (8) -20.3 ± 8.7
Group 2 (n=20) Volume (8) -24.1 ± 8.6
CONTROL Depth (8) -15.3 ± 7.2
(n=20) Volume (8) -15.0 ± 5.2
Table 2: Percentage changes in wrinkle depth and volume over the trial
period from 39 volunteers (mean ± SD).
At 8 weeks the NRFS decreased wrinkle volume by 31.6% more than
the SSID product (p<0.01). Wrinkle depth of the NRFS serum
decreased by 23.4% more so than the SSID product (p=0.0577). e
dierence between the two treatments was most marked from weeks 4
to 8. SSID decreased wrinkle volume by 18.85% over the last 4-week
period while NRFS serum decreased volume by 49.59% (percentage
change in the mean individual percentage changes from the end of
week 4 to the end of week 8) indicating that change was slowing in the
SSID group at a faster rate than that of the NRFS serum. Compared to
Control there was a 55.8% relative reduction wrinkle volume with the
NRFS serum (p<0.01) and a 32.8% decrease in wrinkle depth
(p=0.0123).
Group Parameter Intergroup Improvement p-value
NRFS serum
versus SSID
Wrinkle Depth -32.8 µm 23.4% 0.0577
Wrinkle Volume -0.3 m331.60% 0.0044
NRFS serum
versus
CONTROL
serum
Wrinkle Depth -28.3 µm 32.8% 0.0123
Wrinkle Volume -0.4 m355.80% <.0001
Table 3: Comparisons between treatments aer 8 weeks. a e
Intergroup dierence is the average depth or volume change compared
between the treatments within the same group. A negative result
indicates that change was greater for the NRFS as compared to the
other treatment. b e average improvement of NRFS compared with
the other treatment within the group.
Discussion
In the present paper we report on the eects of GHK-Cu on
synthesis of collagen and elastin, and expression of MMP1, MMP2, as
well as tissue inhibitors of metalloproteinases (TIMP), TIMP1 and
TIMP2, by human adult dermal broblasts (HDFa). ese data are
presented alongside the results of a human clinical trial investigating
eects of GHK-Cu on wrinkle depth and volume.
MMPs/TIMPs expression
Application of GHK-Cu at all the tested concentrations to cultured
human dermal broblasts increased mRNA expression of both MMP1
and MMP2. TIMP1 mRNA expression increased and to a greater
extent than MMP1, suggesting net inhibition of proteolytic activity for
collagens. GHK-Cu, however, did not change TIMP2 signicantly at
0.01 nM, and at higher concentrations decreased expression (p<0.05)
(Figure 1). e ndings of increased MMPs and TIMP1 mRNA
following exposure to GHK-Cu are consistent with the ndings of
Simeon et al. [7] who also examined eects of GHK-Cu on MMPs of
rat broblast cells. e inverse dosage dependent response results from
this study, however, do dier from a study showing that increasing the
free copper concentration increases both MMP1 and TIMP1
expression [25]. Our study with GHK-Cu shows that at higher doses
there is generally little eect or inhibition of both MMPs and their
TIMPs. It is thus likely that GHK-Cu eects that we observed are not
due to free copper.
Irrespective to the dose response, on the other hand, there was clear
correlation between MMPs with their TIMP levels in the GHK-Cu
treated cells; a high concentration of MMP was accompanied with a
high level of its TIMP (Figure 1). A simultaneous increase and decrease
of various MMPs and TIMPs has been previously reported [6]. It is
important to note that TIMPs regulate the proteolytic activity of
MMPs by direct interaction with these enzymes, and not by regulation
at a transcriptional level [26]. It is also important to note that TIMP1
acts against all members of both collagenase and gelatinase classes
[27], thus the no change, or decrease in TIMP2 at higher
concentrations of GHK-Cu, does not necessarily signal a shi in favour
of inammatory changes and increased degradation. e relative
increase in TIMP1 following exposure to GHK-Cu is consistent with a
shi to matrix production and growth, and both TIMP1 and TIMP2
are considered to have growth factor-like functions [27].
Collagen/elastin production
Marquart et al. [5] reported a dosage dependent increase in the
amount of collagen produced by human broblasts incubated with
GHK-Cu. e response peaked at 1 nM with higher concentrations
resulting in less collagen synthesized. In this present study the collagen
levels were above non-treated cells at 96 hours at concentrations of
0.01-100 nM (p<0.05). Increasing GHK-Cu concentrations did not
signicantly increase the response. In contrast, the production of
elastin, measured as α-elastin, was 30% higher than that found in the
untreated cells regardless of the GHK-Cu concentrations (0.01-100
nM) (Table 1).
e increase in collagen production, albeit modest, supports the
conclusion that GHK-Cu stimulates tissue growth and repair. e
increase in elastin similarly supports the conclusion that GHK-Cu
stimulates tissue growth and repair.
Relationship of MMPs/TIMPs expression and collagen/
elastin production
As expected MMPs and TIMPs mRNA expression, and collagen and
elastin production were markedly aected by exposure of the cells to
GHK-Cu. An increase of TIMP1 and TIMP2 suggested an inhibition of
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 5 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
differencea(%) b
proteolytic activity of MMP1 and MMP2 and thus decreased brillar
collagen (collagen) and elastin degradation which is consistent with
the observed increase in collagen/elastin in this study (Table 1).
Surprisingly, the increase in either collagen or elastin appeared to have
little concentration-dependence on GHK-Cu although an inverse
dosage dependent response with TIMP1 and TIMP2 was observed.
More interestingly, although MMP2 increased and TIMP2 decreased
with the treatment with GHK-Cu (0.01-10 nM), which in theory mean
a decreased elastin level in the treated cells, an increase in elastin level
was observed, and again with a little dose-dependence. is means
single factor, either MMP or TIMP cannot determine the level of the
matrix protein.
To fully understand the mRNA results, the ratios of TIMP/MMP
expression were calculated (Table 4). High (>1) and relatively
consistent ratios of TIMP1/MMP1 at all GHK-Cu contractions were
found. is could explain the observation with cellular secretion of
collagen. e low ratio of TIMP2 to MMP2 would predict a shi
towards degradation. Considering however that TIMP1 acts against all
members of the gelatinase classes (MMP2) and that TIMP2 also acts
on MMP2, the ratio of (TIMP1+TIMP2)/MMP2 was used as a
measure of prediction (Table 4).
Ratio GHK-Cu concentration (nM)
0.01 1 100
TIMP1/MMP1 3.76 2.65 3.1
TIMP2/MMP2 0.43 0.37 0.31
(TIMP1+TIMP2)/MMP2 1.72 1.39 1.07
Table 4: TIMP/MMP ratios on HDFa following 24 hour treatment with
GHK-Cu at dierence concentrations (calculated from the mean
values from data in Figure 1).
Clinical study
e eects on MMP and TIMP expression with the associated
increase in collagen and elastin production supported the investigation
of topical GHK-Cu treatment in human volunteers. Due to the
physicochemical properties of GHK-Cu hindering topical absorption,
and given previous studies have evaluated topical GHK-Cu delivery
in
vivo
[12,13], this present study used nano-carriers to facilitate delivery
of GHK-Cu to the dermis. e clinical trial investigated the eect of
GHK-Cu on facial wrinkle volume and depth. e application of
GHK-Cu in nano-carriers (NRFS serum) resulted in a signicant and
improved reduction in facial wrinkle volume and depth compared to
the CONTROL (serum only) and a reduction in wrinkle volume
compared to the SSID, a commercially available product containing a
lipophilic derivative of GHK (Matrixyl 3000®). is conrms the NRFS,
containing GHK-Cu in a carrier system, is more eective than the
SSID product for total wrinkle volume reduction. It also shows that
improvement can be seen with continued use, observed as wrinkle
depth and volume reductions between 0-4 and 4-8 weeks. e NRFS
serum also signicantly outperformed the CONTROL (vehicle only) in
regards to wrinkle volume and depth. is evidence proves that it was
the nano-carrier that impacted signicantly on the total wrinkle
reduction and not the formulation. Of particular importance was that
all participants using NRFS showed a reduction in wrinkle volume and
depth.
Importantly the nano-carrier encapsulated GHK-Cu was well
tolerated and similar to SSID with only one participant reacting to
both. Side-eects of anti-wrinkle agents such as tretinoin are relatively
common and include peeling, dryness and erythema [28]. None of
these were experienced by participants in the GHK-Cu trial,
notwithstanding the aforementioned reaction.
Conclusion
GHK-Cu signicantly increased cellular production of collagen and
elastin by HDFa cells with little concentration dependency (0.01-10
nM). Incubation with the tripeptide also correlated with a relative
higher mRNA expression of the respective TIMP(s) than the respective
MMP in the cells.
Topical application of GHK-Cu with the aid of nano-carrier delivery
systems reduced wrinkle volume to a signicantly greater extent than
the vehicle alone or a commercial product containing Matrixyl 3000®, a
GHK lipophilic derivative.
Acknowledgements
Independent statistical analysis was performed by Jessica McLay
based at the Department of Statistics, e University of Auckland, New
Zealand.
Disclosure
is study was funded by Snowberry New Zealand Ltd in
collaboration with Callaghan Innovation (Grant Number:
ENDU1101.) Snowberry had no involvement in the collection,
analysis, interpretation of data, decision to submit or in the writing of
this article.
References
1. Pickart L, Vasquez-Soltero JM, Margolina A (2012) e human tripeptide
GHK-Cu in prevention of oxidative stress and degenerative conditions of
aging: implications for cognitive health. Oxid Med Cell Longev.
2. Pyo HK, Yoo HG, Won CH, Lee SH, Kang YJ, et al. (2007) e eect of
tripeptide-copper complex on human hair growth in vitro. Arch Pharm
Res 30: 834-839.
3. Pickart L (2008)e human tri-peptide GHK and tissue remodeling. J
Biomater Sci Polym Ed 19: 969-988.
4. Pickart L, Vasquez-Soltero JM, Margolina A (2015) GHK Peptide as a
Natural Modulator of Multiple Cellular Pathways in Skin Regeneration.
BioMed Res Int 648108.
5. Maquart F, Pickart L, Laurent M, Gillery P, Monboisse JC, et al. (1988)
Stimulation of collagen synthesis in broblast cultures by the tripeptide-
copper complex glycyl-L-histidyl-L-lysine-Cu2+. FEBS Lett 238: 343-346.
6. Simeon A, Monier F, Emonard H, Gillery P, Birembaut P, et al. (1999)
Expression and Activation of Matrix Metalloproteinases in Wounds:
Modulation by the Tripeptide–Copper Complex Glycyl-L-Histidyl-L-
Lysine-Cu2&plus. J Invest Dermatol 112: 957-964.
7. Simeon A, Emonard H, Hornebeck W, Maquart FX (2000) e
tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ stimulates
matrix metalloproteinase-2 expression by broblast cultures. Life Sci 67:
2257-2265.
8. Maquart FXP S (1999) Regulation of cell activity by the extracellular
matrix: the concept of matrikines. J Soc Biol 193: 423-428.
9. Van Lint P, Libert C (2007) Chemokine and cytokine processing by
matrix metalloproteinases and its eect on leukocyte migration and
inammation. J Leukoc Biol 82: 1375-1381.
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 6 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
10. Nagase H, Visse R, Murphy G (2006) Structure and function of matrix
metalloproteinases and TIMPs. Cardiovasc Res 69: 562-573.
11. Gill SE, Parks WC (2008) Metalloproteinases and their inhibitors:
regulators of wound healing. Int J Biochem Cell Biol 40: 1334-1347.
12. Leyden J, Stevens T, Finkey M, Barkovic S (2002) Skin care benets of
copper peptide containing facial cream. American Academy of
Dermatology 60th Annual Meeting; New Orleans, Louisiana, USA.
13. Appa Y, Stephens T, Barkovic S, Finkey M (2002) A clinical evaluation of
a copper-peptide-containing liquid foundation and cream concealer
designed for improving skin condition. American Academy of
Dermatology 60th Annual Meeting; New Orleans, Louisiana, USA.
14. Abdulghani A, Sherr A, Shirin S, Solodkina G, Tapia E, et al. (1998)
Eects of topical creams containing vitamin C, a copper-binding peptide
cream and melatonin compared with tretinoin on the ultrastructure of
normal skin-A pilot clinical, histologic, and ultrastructural study. Disease
Management and Clinical Outcomes 1: 136-141.
15. Maquart F, Bellon G, Chaqour B, Wegrowski J, Patt L, et al. (1993) In vivo
stimulation of connective tissue accumulation by the tripeptide-copper
complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds. J
Clin Invest 92: 2368-2376.
16. Watson R, Ogden S, Cotterell L, Bowden J, Bastrilles J, et al. (2009) A
cosmetic ‘antiageing’product improves photoaged skin: a doubleblind,
randomized controlled trial. Br J Dermatol 161: 419-426.
17. Badenhorst T, Svirskis D, Wu Z (2016) Physicochemical characterization
of native glycyl-l-histidyl-l-lysine tripeptide for wound healing and anti-
aging: a preformulation study for dermal delivery. Pharm Dev Technol
21: 152-160.
18. Mitragotri S, Anissimov YG, Bunge AL, Frasch HF, Guy RH, et al. (2011)
Mathematical models of skin permeability: An overview. Int J Pharm 418:
115-129.
19. Kato T, Royce PM (1995) Dierent responses of human and rat dermal
broblasts to L-ascorbic acid 2-phosphate. Biomed Res 16: 191-198.
20. Herrera I, Cisneros J, Maldonado M, Ramrez R, Ortiz-Quintero B, et al.
(2013) Matrix Metalloproteinase (MMP)-1 Induces Lung Alveolar
Epithelial Cell Migration and Proliferation, Protects from Apoptosis, and
Represses Mitochondrial Oxygen Consumption. J Biol Chem 288:
25964-25975.
21. Milia-Argeiti E, Huet E, Labropoulou VT, Mourah S, Fenichel P, et al.
(2012) Imbalance of MMP-2 and MMP-9 expression versus TIMP-1 and
TIMP-2 reects increased invasiveness of human testicular germ cell
tumours. Int J Androl 35: 835-844.
22. Pivarcsi A, Bodai L, Rethi B, Kenderessy-Szabo A, Koreck A, et al. (2003)
Expression and function of Toll-like receptors 2 and 4 in human
keratinocytes. Int Immunol 15: 721-730.
23. Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression
Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method.
Methods 25: 402-408.
24. Biocolor. Soluble Collagen Assay Manual 2012 [cited 2014 11/12/15].
25. Jaspers S, Hopermann H, Sauermann G, Hoppe U, Lunderstadt R, et al.
(1999) Rapid in vivo measurement of the topography of human skin by
active image triangulation using a digital micromirror device. Skin Res
Tech 25: 195-207.
26. Aoki T, Kataoka H, Moriwaki T, Nozaki K, Hashimoto N (2007) Role of
TIMP-1 and TIMP-2 in the progression of cerebral aneurysms. Stroke 38:
2337-2345.
27. Lijnen HR (2002) Matrix Metalloproteinases and Cellular Fibrinolytic
Activity. Biochemistry (Moscow) 67: 92-98.
28. Ellis CN, Weiss JS, Hamilton TA, Headington JT, Zelickson AS, et al.
(1990) Sustained improvement with prolonged topical tretinoin (retinoic
acid) for photoaged skin. J Am Acad Dermatol 23: 629-637.
Citation: Badenhorst T, Svirskis D, Merrilees M, Bolke L, Wu Z (2016) Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin
Production, and Facial Wrinkle Parameters. J Aging Sci 4: 166. doi:10.4172/2329-8847.1000166
Page 7 of 7
J Aging Sci, an open access journal
ISSN:2329-8847
Volume 4 • Issue 3 • 1000166
