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SOFW Journal (2015)
GHK-Cu May Prevent Oxidative Stress in Skin
by Regulating Copper and Modifying Expression of
Numerous Antioxidant Genes Cosmetics (2015)
GHK Peptide as a Natural Modulator of
Multiple Cellular Pathways in Skin Regeneration (2015)
GHK, the Human Skin Remodeling Peptide Induces Anti-Cancer
Expression and DNA Repair Analytical Oncology (2014)
GHK and DNA: Resetting the Human Genome to Health
BioMed Research International (2014)
Avoid Buying Fake Copper Peptides Dangerous


1. GHK, Copper Peptides, and Stem Cells

The primary cause of the diseases of aging is a decline in organ function over time. Up until we reach age 20, tissue and organs are maintained in a fully functional and healthy state. But as we age, repair slows and our organs fail to fulfill their biological role. A key elements of this decline is a reduction in stem cell function.

 

Stem Cells for the skin arise from enlarged hair follicles. The first indication that GHK affects stem cells came from mouse studies where GHK-copper 2+ produced a very strong amplification of hair follicle size. A similar peptide, Ala-His-Lys-copper 2+ produced even stronger actions.
 
  
 

The skin of the mouse to the left was shaved, then treated in three spots with GHK-copper 2+peptides. The result is a much more rapid hair growth (the three circular patches of hair) in the three spots treated with copper peptides.


  
  
  
 

 

 

 

In the microscopic images to the left, the magnifications are identical. The top photo is mouse skin untreated with copper-peptides. The bottom photo is mouse skin treated with copper-peptides. Note the larger hair follicles (the elongated purple columns) in the lower photo, the increased content of subcutaneous fat in the skin (the white material in the center of the skin), and the increased thickness of the skin. When we are young, we have a layer of fat under the skin (part of "baby fat") which is greatly reduced as we age. Hair researchers have noted the accumulation of this fat around healthy follicles that are vigorously growing hair, and its relative lack around dormant follicles, have postulated that these cells serve a supportive function for the hair follicle. It must be emphasized that effects in humans on hair follicle health are not as dramatic. 
  

 

 

 

 

2. Human Marrow Stromal Cells = Mesenchymal Stem Cells = MSC

In 1995, Godet and Marie found that GHK-copper 2+ stimulated the growth of human marrow stromal cells. Today these are called Mesenchymal Stem Cells.

GHK-copper 2+ increased the production of growth factors by MSC. This should increase the effectiveness of medical treatments using MSC.

Godet D1, Marie PJ. Effects of the tripeptide glycyl-L-histidyl-L-lysine copper complex on osteoblastic cell spreading, attachment and phenotype. Cell Mol Biol (Noisy-le-grand). 1995 Dec;41(8):1081-91.

We have studied the effects of the complex Glycyl-L-Histidyl-L-Lysine:Cu (GHK:Cu), the GHK sequence present in the alpha 2 (I) chain of human collagen (Coll I), and bone matrix glycoproteins containing either RGD (fibronectin, FN), or RGD and GHK (Coll I), on the spreading, attachment and markers of the osteoblast phenotype in rat calvaria cells (RC), human trabecular osteoblastic cells (HT) and human marrow stromal cells (HM). Coll I (20 micrograms/ml) and FN (20 micrograms/ml) coating enhanced osteoblastic cell spreading, whereas free GHK:Cu and GHK coating (10(-10)-10(-8) M) had no effect. FN and Coll I, as well as GHK:Cu and GHK, increased the attachment of RC and HT cells. The attachment of both total number of cells and alkaline phosphatase (ALP)-positive osteoblastic cells was increased, showing no preferential effect on cells expressing this early marker of the osteoblast phenotype. In addition, immunocytochemical analysis showed that FN, Coll I and GHK:Cu coating increased both the total number of HM cells and the number of HM cells expressing Coll I or osteocalcin, indicating that GHK:Cu and RGD-containing proteins acted similarly on cells expressing different maturational stages. In contrast to its effect on cell attachment, GHK:Cu coating slightly inhibited the basal and 1,25(OH)2D-induced stimulation of ALP activity or osteocalcin production in rat and human osteoblastic cells. The finding that GHK promotes cell attachment and decreases the phenotype of normal rat and human osteoblastic cells suggests that osteoblasts may interact with free GHK or GHK-containing proteins in the bone matrix.

 

3. Copper Level and Stem Cell Growth or Differentiation

Work by Tony Peled and collegues suggests that low copper in the growth medium promotes stem cell growth and maintenance while higher copper promotes cell differentiation.

The anti-cancer actions of GHK-copper 2+ may be a result of increased copper which forces a differentiation of the cancerous cells. Both GHK-copper and retinoic acid increase tissue regeneration and also act as anti-cancer molecules.

GHK preserved the clonogenic potential of stem cells.

GHK-Cu pushed the stem cells into differentiated cells need by the tissues.

GHK reduced the clonogenic potential of stem cells by 78%. This maintains the stem cell's potential for transformation into more differentiated cells.

GHK-Cu increased cell copper by 2162% above the control value and caused stem cell differentiation

Methods of controlling proliferation and differentiation of stem and progenitor cells, United States Patent: 6,962,698, Peled, Tony, Fibach, Eitan, Treves; Avi, Gamida Cell Ltd. (Jerusalem, IL) and Hadasit Medical Research Services and Development, Ltd. (Jerusalem, IL)

 

4. Generation of Stem Cell Protein Markers

In skin culture keratinocyte-based organ equivalent, GHK-copper 2+ increased keratinocyte replication and the expession of proteins considered to be markers of stem cell activity. To our knowledge, adequate copper is present in all growth media to cause GHK-copper 2+ actions. Attempts to formulate copper-free media failed to reduce reduce free copper below 50 to 300 nanomolar as measured by atomic absorption spectroscopy. Since GHK-copper 2+ is usually maximally active on cells 1-10 nanomolar, the GHK should be able to obtain needed copper from the growth medium.

GHK-copper activated adult stem cells by increasing integrins, P63, and PCNA. This increased keratinocyte proliferation.

Arch Dermatol Res. 2009 Apr;301(4):301-6. Copper-GHK increases integrin expression and p63 positivity by keratinocytes. Kang YA, Choi HR, Na JI, Huh CH, Kim MJ, Youn SW, Kim KH, Park KC. Department of Dermatology, Seoul National University College of Medicine, Yeongeon-dong, Jongno-gu, Seoul, Republic of Korea.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.

Stem cell recovering effect of copper-free GHK in skin.

Pept Sci. 2012 Nov;18(11):685-90. doi: 10.1002/psc.2455. Epub 2012 Sep 28. Choi HR1, Kang YA, Ryoo SJ, Shin JW, Na JI, Huh CH, Park KC.

The peptide Gly-His-Lys (GHK) is a naturally occurring copper(II)-chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper-GHK promotes the survival of basal stem cells in the skin. However, the effects of copper-free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK-treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper-free GHK showed similar effects with copper-GHK. Thus, it can be said that copper-freeGHK can be used in industry to obtain the effects of copper-GHK in vivo. Further study is necessary to explore the relationship between copper-free GHK and copper-GHK.

 

5. GHK Stimulates Growth Activity of Mesemchymal Stem Cells

Stem cell recovering effect of copper-free GHK in skin.

It is likely that the GHK obtained enough trace copper from the growth medium to be effective. We were never able to obtain strong wound healing without additional copper 2+. We also were never able to create a copper-free growth medium because of trace amounts of contaminating copper on the ingredients such as amino acids.

 

But this does not rule out a separate function for copper-free GHK.

Acta Biomater. 2014 May;10(5):1955-64. doi: 10.1016/j.actbio.2014.01.020. Epub 2014 Jan 24. Jose S1, Hughbanks ML1, Binder BY1, Ingavle GC1, Leach JK2.

Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

 


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