GHK–Copper Peptide in Skin Remodeling and Anti

CO S M ET I C S
G H K- CO P P E R P E PT I D E
L. Pickart, A. Margolina*
GHK–Copper Peptide in Skin Remodeling
and Anti-Aging
! GHK-Cu in Skin Remodeling
Although copper-binding tri-peptide glycyl-L-histidyl-L-lysine (GHK-Cu) was first
discovered as an activity that makes old
liver cells behave like young cells, the
following studies soon began to focus on
its wound healing and skin remodeling
activity. These studies provided valuable
insight into GHK-Cu’s molecular actions
and revealed many mechanisms by which
GHK-Cu exhibits anti-aging and tissue
renewing properties. In particular it has
been established that GHK-Cu can increase production of key skin proteins
such as collagen as well as other important components of dermal matrix. In
addition it has been proven that GHK-Cu
modulates the skin remodeling process,
being able to stimulate both breakdown
and synthesis of dermal matrix components. While GHK-Cu is used in most
studies, GHK without copper has been
used in a few instances. But all the available evidence is that GHK exerts its biological effects as its copper complex.
In 1988, Maquart et al. from Université
de Reims Champagne-Ardenne (France)
found that GHK-Cu at concentrations of
10-12 and 10-11 M, maximized at 10-9M was
able to stimulate the synthesis of collagen in fibroblasts. The authors noted
that GHK sequence is present in the alpha 2(I) chain of type I collagen and suggested that in the organism GHK might
be liberated by proteases at the site of a
wound healing (3).
Introduction
S
ince the dawn of time, people have tried to discover the secret of
eternal youth and find a way to reverse aging. Among the most popular ingredients of the elusive elixir of youth sought by ancient mages
and alchemists were herbs, powdered gems, gold, silver and mercury as
well as animal parts and extracts. The most common belief was that the
»essence of youth« can be transferred from young to old people – often in
a form of blood or other body fluids. Today it is known that plasma and
other body fluids (such as saliva) indeed contain a multitude of soluble
growth factors that can enhance reparative and renewing activity of various cells and tissues (1). Of course neither blood nor saliva or other fluids
produced by the human body can be used in cosmetics. Therefore the biological regulators with proven scientific activity that can be purified or produced by chemical synthesis are of particular interest for the cosmetic industry.
One of the anti-aging activities that is present in human plasma and saliva
is a human tri-peptide with an amino acid sequence glycyl-L-histidyl-L-lysine and high affinity for copper (II) (Fig. 1), isolated in 1973 by Dr. L. Pickart.
First discovered as an activity in human plasma albumin that causes old
human liver tissue to synthesize proteins like younger tissue, today GHK-Cu
is widely used as a cosmetic ingredient with confirmed anti-aging and
reparative properties (2).
Fig. 1 The molecular structure of the
tri-peptide GHK-Copper
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In 1992 Maquart et al. used the method
of wound chambers to determine and
quantify components of extracellular
matrix (ECM) produced during wound
healing in rats. GHK increased an accumulation of total ECM proteins, collagen
and glycosaminoglycans as well as increased DNA synthesis. At the same time,
another tri-peptide L-glutamyl-L-histidylL-proline had no significant effect, which
confirmed that the observed stimulation
of skin proteins production was a result
of a specific action of GHK-Cu (4).
P. Ehrlich first observed that GHK simultaneously increases both the synthesis of
new type 1 collagen and the breakdown
of existing collagens in wounds (5). In
1999 Maquart and co-authors demonstrated for the first time that GHK-Cu
regulated skin remodeling process in rats
by modulating activity of different metalloproteinases – enzymes that facilitate
breakdown of proteins of extracellular
matrix (6).
In the next series of experiments, it was
shown that GHK-Cu not only modulates
activity of different matrix metalloproteinases, but also stimulates anti-proteases, maintaining balance between
matrix breakdown and synthesis. The effect was reproduced by the addition of
copper ions, but not by the GHK alone,
which suggests that copper binding activity of GHK is more likely the key mechanism in its tissue remodeling activity
(7). The ability of GHK-Cu to stimulate
both proteases and anti-proteases indicates that it can balance the breakdown
of ECM proteins, preventing excessive
skin damage.
Maquart et al. also demonstrated that
injection of GHK-Cu (2 mg per injection)
into the rat experimental wounds resulted in the increased production of collagen I and glycosaminoglycans (dermatan
sulfate and chondroitin sulfate). Northern blot analysis also showed increased
production of two small proteoglycans
of the dermis – biglycan and decorin. According to the mRNA analysis, the level
of decorin continued to increase until
the end of the GHK treatment (day 22nd).
The ability of GHK-Cu to stimulate decorin
production was also confirmed in fibroblast culture (8).
It was particularly interesting that GHKCu increased decorin, since this proteo-
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glycan functions by regulating assembly
of collagen fibrils, preventing scar formation and decreasing the level of TGFbeta, which is known to increase scarring
(9-10). In addition GHK-Cu has been
shown to decrease TGF-beta production
in serum-free fibroblast culture (human
cells obtained during facial surgery) (11).
This may be explained through the increased decorin level, however it is also
possible that GHK-Cu itself inhibits TGFbeta.
Also, GHK-Cu was found to attract immune and endothelial cells to the site of
injury, which is another explanation of
its wound healing promoting activity.
The most recent study (2007) of wound
healing activity of GHK-Cu was conducted in Taiwan. Huang et al. evaluated the
effect of GHK alone or in combination
with LED irradiation (light emitting diode
irradiation, 625-635 nm) on human fibroblasts. Combined GHK and LED treatment resulted in 12.5-fold increase in
cell viability, 230% increase in basic fibroblast growth factor (bFGF) production, and 70% increase in collagen I mRNA production compared with the LED
irradiation alone (13). Since fibroblasts
are the key cells in skin reparative and renewal processes, the ability of GHK-Cu
to support these cells increasing their
functional activity may in part explain its
wound healing and skin rejuvenating activity.
GHK peptide belongs to a family of biologically active regulators that are naturally present in extracellular matrix (ECM)
and released from it after injury. These
»first emergency response« molecules are
called matrikines (14). In addition to being a part of collagen molecule, GHK sequence is also found in a glycoprotein
SPARC that is produced by endothelial
cells in the site of injury. After an injury
GHK released from a SPARC molecule by
proteolysis (15).
Therefore GHK-Cu is not only naturally
present in skin, but it is also liberated
from skin’s proteins after stress or injury.
Considering its ability to stimulate the
synthesis of key skin proteins as well as
to increase functionality of skin fibroblasts, it should be quite effective as a
wound healing agent. And indeed, animal experiments fully support this statement.
! GHK Improves Wound Healing
in Animals
A series of in vivo experiments confirmed
wound healing activity of GHK-copper.
In rabbits, GHK facilitated wound healing, causing better wound contraction,
faster development of granular tissue
and improved vessel growth (16). Also
GHK alone and in combination with high
dose helium neon laser stimulated granulation, increased the formation of new
blood vessels and elevated the level of
antioxidant enzymes in the dermal wounds
in rabbits (17).
GHK-Cu was used to create a novel wound
dressing – collagen membrane with incorporated biotinylated GHK. This material has been shown to significantly improve wound healing processes in rats
when compared to non-treated wounds
and to wounds treated with collagen
matrix alone. Collagen dressing with incorporated GHK stimulated wound contraction and cell proliferation, as well as
increased expression of antioxidant enzymes (18).
The most important finding was GHK’s
ability to improve wound healing in difficult to heal wounds, such as diabetic
wounds in rats. GHK-Cu treatment resulted in faster wound contraction and
epithelization, higher level of antioxidants such as glutathione and ascorbic
acid, increased synthesis of collagen, and
activation of fibroblasts and mast cells
(19). Also GHK-Cu improved healing of
ischemic open wounds in rats. Wounds
displayed faster healing, decreased concentration of metalloproteinases 2 and
9 as well as of TNF-alpha (a major inflammatory cytokine) compared with
vehicle alone or with untreated wounds
(20). GHK-Cu triggered an accelerated
healing of the injury after mild thermal
burns to the backs of pigs. The rate of the
burn wound healing gave a linear, doseresponse up to 1% ionic copper in the
cream. In pigs, punch biopsies were used
to create wounds. The defects were filled
with graded amounts of GHK-Cu in a
cream or control substances. In a dose
dependent manner, GHK-Cu stimulated
wound healing and collagen synthesis.
The effect was highly localized to the
immediate skin area that was treated
(21).
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G H K- CO P P E R P E PT I D E
Animal experiments confirmed that GHKcopper has great potential to improve
skin conditions. It speeds up skin repair,
improves circulation, strengthens antioxidant defense, and increases production of skin proteins. All these qualities
are highly desirable in cosmetics. The
ability of GHK-Cu to improve difficultto-heal wounds can be helpful in managing skin healing after plastic surgery,
especially in advanced age patients and
in patients with underlying health conditions.
! Cosmetic Use of GHK-Cu
Today peptide based cosmetic products
continue gaining popularity. However,
not all peptides that are used in today’s
cosmetic products have enough scientific data confirming their efficacy. Also
the main problem with peptide-based
cosmetics is that most peptides cannot
pass through stratum corneum and
therefore cannot reach viable layers of
epidermis. Since peptides are used for
their cell regulatory activity, inability to
permeate stratum corneum is a serious
handicap.
Mazurowska et al. demonstrated that
GHK-Cu is able to pass through the lipid
barrier of the stratum corneum and to
reach epidermal cells (22). Moreover,
GHK-Cu complex appears to be the only
peptide-copper complex that can penetrate stratum corneum membranes (23).
At present, GHK-Cu undoubtedly has
more scientific support data than any
peptide used in today’s cosmetic practice
(24). Its efficacy was confirmed in several placebo-controlled independent trials.
A study of 20 women compared the
skin's production of collagen after applying creams containing GHK-Cu, vitamin C, or retinoic acid to thighs daily for
one month. New collagen production
was determined by skin biopsy samples
using immunohistological techniques.
After one month, GHK-Cu increased collagen in 70% of those treated against
50% treated with vitamin C and 40%
treated with retinoic acid (25).
A GHK-Cu facial cream reduced visible
signs of aging after 12 weeks of application to the facial skin of 71 women with
mild to advanced signs of photoaging.
The cream improved skin laxity, clarity,
4
and appearance, reduced fine lines and
the depth of wrinkles, and increased skin
density and thickness comparing with
placebo (26).
A GHK-Cu eye cream, tested on 41 women
for twelve weeks with mild to advanced
photodamage, was compared to a placebo control and an eye cream containing
vitamin K. The GHK-Cu cream performed
better than both controls in terms of reducing lines and wrinkles, improving
overall appearance, and increasing skin
density and thickness (27).
In another 12 week facial study of 67
women between 50-59 years with mild
to advanced photodamage, a GHK-Cu
cream was applied twice daily and improved skin laxity, clarity, firmness and
appearance, reduced fine lines, coarse
wrinkles and mottled hyperpigmentation, and increased skin density and
thickness. The result was assessed visually by a trained technician (wrinkles, pigmentation, laxity, roughness, overall appearance) as well as using ballistometer
(firmness of the skin) and ultrasound
(skin density). The GHK-Cu cream also
strongly stimulated dermal keratinocyte
proliferation as determined by histological analysis of biopsies. At the same time,
GHK-containing cream has proved to be
very safe. GHK-Cu complex at 20x use
level was proved to be non-allergenic. It
also did not produce eye irritation (28).
! Stimulation of Hair Growth
From the very first experiments with
GHK-Cu, we observed exceptionally large
hair follicles developing at the periphery
of wounds treated with GHK-Cu. Today
it is known that hair follicles are the important source of stem cells that actively participate in skin repair and are capable of differentiating into many skin
cell lineages (29).
The hair growth stimulating effect of
GHK-Cu was confirmed in the following
experiment. A 25 days old C3H mouse
was shaved and injected in three spots
with GHK-Cu. According to the radioisotope analysis, GHK-Cu was present at the
injection site for about 30 sec, before
clearing from the area. However, this
brief exposure was enough to stimulate
hair growth. In 12 days there was strong
hair growth stimulation at the injection
site (Fig. 2) (30).
In fuzzy rats a copper binding peptide
PC1031, a structural analog of GHK
(ghkvfv-copper complex) affected hair
growth with its effect comparable to
that of minoxidil. Both 5% minoxidil and
5% PC1031 almost doubled follicle size
after 3-4 months of treatment, and
caused 80% increase in the number of
anagen hair follicles. An increased DNA
synthesis and cell proliferation was confirmed in the enlarged hair follicles (31).
Fig. 2 A 25 day-old mouse was shaved and injected intradermally in three spots
with GHK-Cu. Twelve days later, there was a very strong stimulation of hair growth
at the injection sites
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The mechanism of GHK-Cu induced hair
growth was investigated in experiments
with GHK-Cu analog – AHK (L-alanyl-Lhistidyl-L-lysine-Cu2+). It was found that
AHK-Cu stimulated dermal papilla cells
(DPCs) – specialized fibroblasts important in the morphogenesis and growth of
hair follicles. AHK caused elongation of
hair follicles, stimulated proliferation of
DPCs and prevented their apoptosis.
Today, analogs of GHK-Cu with hydrophobic amino-acids are used to enhance hair growth in the commercial
product Tricomin (Procyte Corp). Also a
product GraftCyte (Procyte Corp) is used
to increase success of hair transplants
with clinically confirmed efficiency (33).
! GHK’s Effect on Skin Stem Cells
The ability of GHK-Cu to rejuvenate aging skin and to stimulate hair growth indicates its important role in skin and hair
renewal process. Recent studies revealed
one possible mechanism of this rejuvenating action. It has been discovered that
GHK is able to restore proliferative potential of adult stem cells residing in skin.
In 2009, a group of researchers from the
Seoul National University (Republic of
Korea) used multilayered skin equivalent
(SE) model to demonstrate that GHKcopper is able to increase proliferative
potential of basal human keratinocytes
as well as expression of epidermal stem
cell markers.
Epidermal stem cells are slow cycling
cells in a basal layer that maintain nondifferentiated state through the entire
life time of an organism. They give rise
to transient stem cells that in turn differentiate into keratinocytes. In case of
an injury, epidermal stem cells are able
to differentiate into all types of cells
found in the skin, including fibroblasts,
melanocytes, endothelial cells etc.
Although not all characteristics of epidermal stem cells are known, their main
features are believed to be a cuboidal
shape, high integrin expression and an
expression of p63 protein – a member of
p53 family – that is often used as a stem
cell marker. Loss of proliferative poten-
tial of skin stem cells is associated with
flattened shape and decreased expression both integrins and p63. Integrins are
surface proteins that play an important
role in maintaining an attachment of
stem cells to the basement membrane. It
is believed that such attachment is required for maintaining immature, noncommitted state of stem cells – their
»stemness«.
In cell cultures GHK copper (0.1-10 microM) stimulated proliferation of keratinocytes in a dose dependent manner
and was not toxic in these concentrations. An addition of GHK-copper resulted in noticeable changes in epidermal
basal cells. Their integrins and p63 expression markedly increased and their
shape became more cuboidal, as is characteristic for stem cells. The authors concluded that GHK-copper is able to maintain »stemness« of basal keratinocytes as
well as to revive their proliferative potential (34).
This discovery is especially important in
the light of the recent findings that suggest that age-related decline of the pro-
Fig. 3 GHK-Cu effects on aged skin
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liferative capacity of stem cells may be
due to disrupted regulation from the
aged environment rather than to stem
cells senescence. In contrast to the prevalent theory, according to which all human cells have limited proliferative capacity of approximately 50 divisions (so
called Hayflick limit), in some renewing
tissues stem cells have been shown to
undergo more than 1000 divisions without showing signs of senescence (35).
New findings suggest that epidermal
stem cells may be intrinsically aging resistant and that local microenvironment,
rather than cellular senescence, is responsible for their functional decline (36).
GHK’s ability to revive proliferative potential of epidermal stem cells makes it
a powerful player in skin repair and rejuvenation process (Fig 3).
! GHK-Cu Restores Function in
Damaged Cells
A recent study showed GHK-Cu's ability
to restore function of irradiated fibroblasts to that of intact cells. The researchers used cultured human fibroblasts obtained from cervical skin that was
either intact or exposed to radioactive
treatment (5000 rad). GHK copper (109
M) was added in a serum free medium
directly to the cell culture. An equivalent
amount of plain serum-free medium was
added to control cells (37).
Although irradiated fibroblasts survived
and replicated in the cell culture, their
growth dynamics were markedly different from that of intact cells. The growth
of the irradiated cells was especially delayed at 24 and 48 hour measurements.
However, the irradiated fibroblasts treated with GHK showed much faster growth,
similar to the normal (non-irradiated
control cells). In addition GHK-treated
irradiated fibroblasts showed increased
production of growth factors bFGF and
VEGF, which was significantly higher
than that of irradiated and intact control cells (37).
Fibroblasts are central cells in both wound
healing and tissue renewal processes. They
not only synthesize different components of ECM, but also produce a number of growth factors that regulate cell
migration and proliferation, angiogene-
6
sis, epithelialization etc. However, fibroblasts are sensitive cells that can be
damaged by many factors. The fact that
GHK-Cu restored damaged fibroblasts’
activity up to a level to that of normal,
non-irradiated fibroblasts, opens new
possibilities in enhancing fibroblasts’
function in aged skin.
! GHK-Cu Suppresses Cancer
This area of GHK-copper’s activity may
belong to medicine rather than cosmetology, however it sheds an important
light on the protective and reparative
role of GHK-Cu in the organism.
In the study published in 2010 by Hong Y.
et al. (Department of Colorectal Surgery,
Singapore General Hospital, Singapore)
GHK-Cu was selected out of 1309 biologically active substances as an activity
that was able to reverse expression of
certain genes involved in metastatic
spreading of colon cancer. Another substance that produced such effect was
plant alkaloid securinine. GHK-Cu was
not toxic and produced effect at a very
low concentration - 1mkM (securinine
was active at 18 microM) (38).
This research is the first that reports direct anti-cancer activity of GHK-copper.
However, there is also indirect evidence
suggesting an important role of GHKcopper in preventing cancer.
First of all, GHK-Cu can exhibit anti-tumor activity by increasing decorin (10).
It has been shown that stimulation of
decorin expression leads to the regression of certain tumors, such as gliomas.
Decorin also was found to inhibit growth
of various tumors and to prevent metastasis of breast cancer (39). One of the important qualities of decorin is its ability
to inactivate TGF-beta. It is known that
TGF-beta helps the tumor to escape the
action of the host’s immune system by
suppressing immune response, causing
apoptosis of immune cells and modulating the activity of growth factors (40).
GHK-Cu is also proved to decrease TGFbeta, although this effect may be secondary – due to the increased decorin
level.
Another contribution of GHK-copper into anti-tumor defense of an organism as
well as into prevention of other age re-
lated degenerative conditions is its antioxidant and anti-inflammatory action.
! Anti-inflammatory and
Antioxidant Action
It is known that inflammation is always
accompanied by increased oxidative damage. One reason is that immune cells use
reactive oxygen species (ROS) and other
powerful oxidants (such as hypochlorite
and peroxinitrite) to combat the bacteria. Since free radicals play an important
role in the aging process, antioxidant
and anti-inflammatory ingredients are
often included in anti-aging cosmetic
formulations (41).
It was found that GHK-Cu suppresses inflammation by lowering the level of acute
phase inflammatory cytokines such as
TGF-beta and TNF-alpha (42). GHK also
reduces oxidative damage by modulating iron levels (43) and by quenching
toxic products of fatty acids lipid peroxidation (44-45). Since lipid peroxidation
plays an important role in skin aging and
UV-induced skin damage, the ability of
GHK to form complexes and sequester
toxic by-products of lipid peroxidation
has a protective effect against skin aging and photo-damage. One of the possible application of GHK-Cu is sun-protective cosmetics. It is known that many
synthetic UV-filters can generate free
radicals and increase oxidative damage
to the skin. An addition of GHK-Cu to
sun-protective cosmetics may decrease
the level of harmful by-products of lipid
peroxidation, reduce inflammation and
skin damage.
! How Does GHK-Cu Work?
GHK tri-peptide has a strong affinity for
copper and more often exists in a form
of GHK-Cu. On the basis of available data it was proposed that GHK-Cu might
function by modulating copper intake
into cells (46).
It is now well established that the biological significance of GHK-Cu primarily
results from its unique relationship with
copper. Since copper ions are used by
more than a dozen enzymes involved in
different biological processes in the cell,
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CO S M ET I C S
G H K- CO P P E R P E PT I D E
supplying the tissues with copper can
improve many aspects of tissue metabolism including antioxidant defense, tissue repair, oxygenation etc. Among enzymes that use copper are an antioxidant enzyme superoxide dismutase
(SOD), lysyl oxidase is required for connective tissue formation, tyrosinaze responsible for melanin synthesis and
many others (47).
Another important function of copper is
signaling. For example, low tissue copper
prompts proliferation of stem cells, while
sufficient tissue copper is required for
stem cell differentiation into cells needed for tissue repair (48). An inadequate
level of copper can prevent stem cells
from differentiation and hamper tissue
repair (49). By modulating copper level
GHK-Cu may act as a »switch« that curbs
inflammation and prompts skin healing
and remodeling process (Fig. 4). Small
size and mobility allow GHK-copper easy
access to molecular receptors and speedy
travel in the extracellular space.
Fig. 5 GHK-Cu and »The Copper switch«.
Fig. 6 Proposed mechanism of action of GHK-Cu
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However, it would be a mistake to view
GHK-Cu simply as a copper transporting
protein. As it has been discovered recently, its biological activity goes well
beyond copper binding.
A number of studies show that tri-peptide is able to bind not only with copper,
but also with a variety of biological molecules as well as promote cell attachment to different substrates.
Rabenstein et al. demonstrated that GHK
is able to form complexes with heparin
(50). It is known that heparin shares common characteristics with heparin sulfate
– one of the main GAGs of the epidermal basement membrane (51). The ability of GHK to bind with heparin indicates
its ability to bind also with heparin sulfate, and therefore – with basement membrane and ECM. Since GHK also can bind
with cellular receptor, its ability to bind
with ECM allows it to facilitate cell adhesion to ECM structures, stimulating
their migrating and differentiating (52)
(Fig. 5).
By modulating copper level and facilitating cellular interaction with extracellular matrix, GHK-Cu is able to exhibit
broad reparative, protective and rejuvenating activity acting as a natural skin
wellness and age reversal agent.
! Conclusion
Human tri-peptide GHK-Cu is a natural
skin wellness molecule that exhibits a
broad range of reparative, anti-aging,
and protective actions. By regulating
processes of skin renewal and repair, it
ensures not only fast healing of dermal
wounds, but also exhibits rejuvenating
action by increasing water-holding molecules in dermal matrix, stimulating collagen synthesis, balancing the action of
skin proteases and reducing oxidative
damage. Particularly interesting is its
ability to maintain epidermal stem cells
as well as to restore viability of damaged
skin cells such as irradiated fibroblasts.
Since fibroblasts and adult stem cells
play a crucial role in skin renewal and
wound healing process, GHK-Cu presents
itself as a natural skin renewing, repairing and rejuvenating ingredient.
Possible applications of cosmetics containing GHK-copper include:
8
1. Anti-aging topical formulations – slowing down aging, reducing wrinkles,
improving skin complexion, reducing
unwanted pigmentation, improving
skin structure.
(8)
Siméon A, Wegrowski Y, Bontemps Y, Maquart FX. Expression of glycosaminoglycans
and small proteoglycans in wounds: modulation by the tripeptide-copper complex glycylL-histidyl-L-lysine-Cu(2+).J Invest Dermatol.
2000;115(6):962-8
2. Cosmetic formulations intended to be
used before and after aggressive cosmetic procedures and plastic surgery –
speeding up skin healing, reducing
the risk of side effect, improving outcome. Such formulations would be
especially valuable for patients of advanced age or with underlying health
conditions.
3. Topical formulations for reducing inflammation and redness associated
with various cosmetic manipulations.
(9)
Sayani K, Dodd CM, Nedelec B, Shen YJ, Ghahary A, Tredget EE, Scott PG. Delayed appearance of decorin in healing burn scars.
Histopathology 200; 36(3): 262-272
4. Sun-protective formulations.
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Address of the authors’:
Loren Pickart PhD
Anna Margolina PhD
Skin Biology, Inc
4122 Factoria Boulevard - Suite 200
Bellevue, WA 98006
Email:
[email protected]
[email protected]
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SOFW-Journal | 136 | 6-2010
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