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. 2018 Feb 21;15(4):538–546. doi: 10.1111/iwj.12895

Growth hormone‐releasing peptide 6 prevents cutaneous hypertrophic scarring: early mechanistic data from a proteome study

Maday Fernández‐Mayola 1, Lázaro Betancourt 2, Alicia Molina‐Kautzman 1, Sucel Palomares 2, Yssel Mendoza‐Marí 1,✉, Dayana Ugarte‐Moreno 3, Ana Aguilera‐Barreto 4, Yilian Bermúdez‐Álvarez 4, Vladimir Besada 2, Luis J González 2, Ariana García‐Ojalvo 1, Ana J Mir‐Benítez 5, Aleida Urquiza‐Rodríguez 6, Jorge Berlanga‐Acosta 1
PMCID: PMC7949743  PMID: 29464859

Abstract

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing with excessive extracellular matrix (ECM) deposition. Current therapies still fall short and cause undesired effects. We aimed to thoroughly evaluate the ability of growth hormone releasing peptide 6 (GHRP6) to both prevent and reverse cutaneous fibrosis and to acquire the earliest proteome data supporting GHRP6's acute impact on aesthetic wound healing. Two independent sets of experiments addressing prevention and reversion effects were conducted on the classic HTS model in rabbits. In the prevention approach, the wounds were assigned to topically receive GHRP6, triamcinolone acetonide (TA), or vehicle (1% sodium carboxy methylcellulose [CMC]) from day 1 to day 30 post‐wounding. The reversion scheme was based on the infiltration of either GHRP6 or sterile saline in mature HTS for 4 consecutive weeks. The incidence and appearance of HTS were systematically monitored. The sub‐epidermal fibrotic core area of HTS was ultrasonographically determined, and the scar elevation index was calculated on haematoxylin/eosin‐stained, microscopic digitised images. Tissue samples were collected for proteomics after 1 hour of HTS induction and treatment with either GHRP6 or vehicle. GHRP6 prevented the onset of HTS without the untoward reactions induced by the first‐line treatment triamcinolone acetonide (TA); however, it failed to significantly reverse mature HTS. The preliminary proteomic study suggests that the anti‐fibrotic preventing effect exerted by GHRP6 depends on different pathways involved in lipid metabolism, cytoskeleton arrangements, epidermal cells’ differentiation, and ECM dynamics. These results enlighten the potential success of GHRP6 as one of the incoming alternatives for HTS prevention.

Keywords: fibrosis, GHRP6, hypertrophic scar, keloid, wound

1. INTRODUCTION

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing processes characterised by an exaggerated fibroproliferative response caused by deep dermal injury. They generally appear as red, raised, rigid scars that can cause pain, pruritus, poor joint mobility, and cosmetic deformities, affecting the patient's quality of life.1, 2 The normal wound‐healing physiological process depends on the fine tuning of the balance between extracellular matrix (ECM) deposition and degradation. Both excessive ECM production as a result of hyperactivation of fibroblasts and myofibroblasts and poor ECM degradation and remodelling because of impaired matrix metalloproteinase expression can lead to HTS formation.2 Even though several attempts have been made to elucidate the molecular mechanisms leading to HTS, the pathogenesis of excessive scarring is still poorly understood. Current therapies targeting this clinical niche are still far from the desired outcome as either surgical or medical scopes, as well as combinations such as surgical removal followed by intralesional steroid therapy or cryotherapy, still have high reoccurrence rates. Corticosteroid therapy is likely the first‐line choice for keloid prevention and treatment at primary health care levels.3 TA suspension (10‐40 mg/mL) is the most broadly used corticosteroid for abnormal scar treatment. Even when corticosteroids have been shown to be relatively effective for excessive scarring treatment, they remain a troublesome therapy with common side effects, such as skin and subcutaneous fat atrophy, telangiectasias, hypopigmentation, rebound effects, and ineffectiveness.4 The present treatment scenario has opened the door for a wide range of novel therapeutic options still under research; nevertheless, their clinical efficacy and safety still have to be proved.3

Growth hormone‐releasing peptide 6 (GHRP6) is a ghrelin‐like GH secretagogue that has shown cyto‐protective and pro‐survival effects in multiple experimental scenarios.5, 6, 7 Previous findings from our group demonstrate GHRP6's capability to both prevent and reverse liver fibrosis sustained by a significant reduction of pro‐fibrogenic genes as TGF‐β and CTGF8 encouraged us to assess GHRP6's potential to prevent exuberant scarring in the classic rabbit model of HTS induction. We recently published the first preliminary evidence that, as expected, GHRP6 did prevent the onset of experimental HTS.9

Here, we thoroughly evaluated GHRP6 consistency in both the prevention and reversion of HTS in the experimental rabbit model. For the first time, we used ultrasonographic procedures to characterise experimental HTS, and we acquired the earliest relevant mechanistic proteome data supporting the anti‐fibrogenic effects of GHRP6.

2. MATERIALS AND METHODS

2.1. Ethics

The animal experiments described here were conducted according to the Program for the Use and Management of Laboratory Animals for Experimental Purposes and for the Control of Biotechnological Products set by the Animal Welfare Committee of the Center for Genetic Engineering and Biotechnology, Havana, Cuba (Ed. 06/2016).

2.2. Formulations

GHRP6 hexapeptide (His‐d(Trp)‐Ala‐Trp‐d(Phe)‐Lys‐conh2) was purchased from BCN Peptides S.A., Barcelona, Spain. The GHRP6‐based parenteral formulation used in the reversion scheme was a sterile, pyrogen‐free, lyophilised composition containing dihydrate α,α‐trehalose and l (+) tartaric acid.

TA (40 mg/mL, Pharmaceutical Laboratory “Julio Trigo López”, Medicuba Group, Havana, Cuba) was kindly donated by one of the authors.

2.3. Surgical procedure for HTS induction

New Zealand male rabbits (4.3‐4.5 kg) were used to assess the anti‐fibrotic effect of GHRP6. Rabbits were anaesthetised with intramuscular ketamine (60 mg/kg) and xylazine (5 mg/kg) before starting surgical procedures. Three to four wounds were inflicted on the ventral side of each ear using 6‐mm punch sterile biotomes (Acuderm, Ft. Lauderdale, FL USA) as described.10, 11 Briefly, wounds were inflicted avoiding the ears’ central artery and marginal veins. Perichondrium was carefully removed in order to ensure exuberant scarring.

2.4. Prevention experimental scheme

After surgical HTS induction, 24 animals were randomly assigned to 3 experimental groups as follows:

  • Vehicle: 1% sodium CMC (N = 8 rabbits/48 wounds).

  • GHRP6: GHRP6 400 μg/mL in 1% CMC (N = 8 rabbits/46 wounds).

  • TA: Triamcinolone acetonide 2 mg/mL (N = 8 rabbits/50 wounds).

Treatments were topically applied accordingly using 1‐mL sterile, disposable syringes. Treatment sessions began immediately after surgery and continued thereafter until day 30, when most of the wounds had already achieved reepithelialisation. The incidence of firm, protruded nodules with a nipple‐like appearance arising in resurfaced wounds was registered daily from day 1 until day 25 post‐wounding, in addition to the non‐epithelialised wounds. HTS detected were characterised by ultrasonographic procedures that allowed size determination. Animals remained in observation for another 20 days after treatment completion, until euthanasia and sample processing for haematoxylin/eosin (H/E) staining.

2.5. Reversion experimental scheme

After surgical HTS induction, the wounds of 10 animals were macroscopically followed during 50 days until a steady mature HTS phenotype was established. Exuberant scars were counted and characterised ultrasonographically at day 50 in order to randomly assign animals to the following experimental groups:

  • Vehicle: sterile saline solution (N = 5 rabbits/25 wounds).

  • GHRP6: GHRP6‐based parental formulation 400 μg/250 μL in sterile saline solution (N = 5 rabbits/29 wounds).

HTS were infiltrated daily accordingly for 4 consecutive weeks (except weekends) using 1‐mL sterile, disposable syringes with 29 1/2 needles. Once treatment concluded, HTS were characterised by ultrasonography. The animals were terminated on day 78 post‐injury, and the scars were resected in block and processed for H/E staining.

2.6. Ultrasonographic characterisation of HTS

Following different probing sessions, we determined and estimated the area of each fibrotic core located between the epidermis and perichondrium of the rabbit's ears. Ultrasonographic images were collected using a Mindray DC‐8 Diagnostic Ultrasound System coupled with a soft‐tissue 7.5–12 MHz probe (Mindray DS Inc. Mahwah, NJ, USA). Images corresponding to each fibrotic core detected were properly delimited and frozen, and the resulting data were averaged.

2.7. Histopathological analysis

After euthanasia (anaesthesia overdose), the wounds were collected in block with a margin of intact skin to ensure proper embedding orientation. The tissue fragments were longitudinally bisected along the largest point of nodular growth. Both hemi‐sections were fixed in 10% neutral buffered formaldehyde and processed for H/E staining. Scar overgrowth was measured using the previously described scar elevation index (SEI) based on the cross‐sectional scar area to the area of tissue excised to induce the wound.12 Three blinded researchers measured the sections using the ImageJ software package (version 1.46 for Windows) (Bethesda, MD; USA).

2.8. Proteomic experiment and bioinformatic analysis

After surgical HTS induction, 10 animals were randomly allocated to the following experimental groups:

  • Vehicle: 1% sodium CMC (N = 5 rabbits/40 wounds).

  • GHRP6: GHRP6 400 μg/mL in 1% CMC (N = 5 rabbits/40 wounds).

Animals received 1 topical administration of either GHRP6 or vehicle accordingly and remained in quarantine for 1 hour until euthanasia and sampling. Wounds and adjacent tissue were collected using 8‐mm diameter sterile punch biotomes (Acuderm, USA). Samples were extensively washed in phosphate‐buffered saline solution containing a protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN, USA), pooled accordingly, and homogenised for 25 minutes close to 0°C. Then, they were centrifuged at 16 000g for 1 hour at 4°C and treated as described.13 Briefly, 300 μg of total proteins were reduced, carbamidomethylated, and digested in tandem with lysyl‐endopeptidase and trypsin at an enzyme/substrate ratio of 1:50 (wt/wt). For a proper relative quantification of proteins from each evaluated condition, the primary amine groups of proteolytic peptides were derivatised with non‐labelled or 13C2‐labelled N‐acetyl N‐hydroxysuccinimide ester accordingly. Peptide pools were collected from a strong cation exchange SCX Biobasic column (4.6 × 250 mm, 300 Å, Thermo Scientific, Waltham, MA, USA) and analysed by LC–MS/MS through an Easy‐column 75 μm × 100 mm column (Thermo Scientific) coupled with a LTQ‐Orbitrap Velos Pro mass spectrometer (Thermo Scientific, USA). The 400 to 2000, 400 to 900 (low m/z), and 890 to 1600 (high m/z) mass ranges were acquired using the gas‐phase fractionation method.14 Three technical replicates were obtained for each chromatographic run, and higher‐energy collision dissociation MS/MS spectra were obtained for the 15 most intense signals. Protein identification and quantitation were performed with MASCOT Distiller 2.5.1.0 and MASCOT 2.3.02 considering the taxonomy of Oryctolagus cuniculus from nrNCBI sequence database. A false discovery rate lower than 1% at the peptide level was accepted for protein identification. Relative quantification was performed by comparing the intensities of the light (CH3CO─) and heavy (13CH3 13CO─) components of each peptide.

Proteins differentially modulated by GHRP6 were analysed using 2 main groups of bioinformatic tools: enrichment analysis and text mining. The Gene Ontology (GO) biological process, molecular function, and the cellular component, together with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses, were carried out using the DAVID (http://david.abcc.ncifcrf.gov) enrichment tool. Text mining was applied using Chillibot (http://www.chilibot.net/) searching for interactions between the proteins and main topics related to keloid pathogenesis. The results were curated by false positives considering bibliographic references linked by the tool. Literature information was also retrieved and analysed with GoPubMed (http://www.gopubmed.org/web/gopubmed/). The biologically relevant annotations were selected using the enrichment analysis results, in combination with UNIPROT (http://www.uniprot.org/) and literature information. For the analysis of uncharacterised proteins, a similarity sequence‐based search approach was applied, using the blastp tool available on the NCBI site (http://blast.ncbi.nlm.nih.gov/). The query sequences were obtained from the information linked to Uniprot accession numbers, and the searching algorithms were constrained to the human species. Based on similarity scores and findings redundancies, human homologues proteins were selected and studied using the procedure described above.

2.9. Statistics

Statistical analyses were carried out using GraphPad Prism software 6.01 (La Jolla, CA, USA). Experimental datasets were first evaluated for a Gaussian distribution using the D'Agostino and Pearson omnibus normality test. Comparisons between groups were assessed using either ordinary one‐way ANOVA followed by Tukey's multiple comparison test when datasets fitted normal distribution or Kruskal‐Wallis test followed by Dunn's multiple comparisons test for datasets without Gaussian distribution. A P value <.05 was considered statistically significant.

Data from the proteomic experiment were analysed using Minitab statistical software (version 17.1.0) (State College, PA, USA). The statistical processing included the normality test for peptides’ fold‐change values distribution. A histogram with all the heavy/light (H/L) peptide ratios was obtained and tested for normal distribution. Proteins with a P value lower than 0.1 were considered to be differentially expressed, either being up‐ or down‐regulated. Proteins containing one or more peptides with an H/L ratio higher than 2.19 or lower than 0.45 were considered to be differentially modulated by GHRP6.

3. RESULTS

3.1. Prevention experimental scheme

3.1.1. Macroscopic findings

GHRP6 treatment prevented the onset of detectable HTS as well as the first‐line treatment TA (Figure 1a). However, TA treatment was demonstrated to disrupt the natural course of the wound‐healing process as several wounds remained non‐reepithelialised or even ulcerated compared with their GHRP6‐ and vehicle‐treated counterparts in the period from day 14 to day 25 post‐wounding (Figures 1b and 2c).

Figure 1.

Figure 1

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Prevention experimental scheme. Hypertrophic scars (HTS) detected by palpation (A) and non‐reepithelialised wounds (B) from day 14 to day 25 post‐injury (both expressed as a percentage of the total number of lesions induced). Kruskal‐Wallis test followed by Dunn's multiple comparisons test. *P < .05, **P< .01, NS, no significance; TA, triamcinolone acetonide

Figure 2.

Figure 2

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Prevention experimental scheme. Aesthetic appearance of vehicle‐ (A), GHRP6‐ (B), and triamcinolone‐ acetonide (C)‐treated lesions along the examination period. Black arrows indicate representative lesions

During the examination period, most of both GHRP6‐ and TA‐treated wounds evolved to normochromic flat appearance scars that were negative at palpation. In contrast, the wounds treated with the CMC vehicle gel evolved to be nipple shape‐like, elevated, and hyperaemic nodules of firm consistency (Figure 2). Even so, it was possible to detect GHRP6‐ and TA‐treated, non‐responsive wounds (around 10%) that evolved to an exuberant and hyperchromic phenotype irrespective of the treatments. The HTS derived from these non‐responsive wounds exhibited similar macroscopic aesthetic appearances to those of the CMC vehicle group (not shown).

3.1.2. Ultrasonographic characterisation of HTS

HTS characterisation by ultrasonographic procedures allowed the determination of HTS size. The fibrotic cores, located between the epidermis and perichondrium of the rabbit's ears, appeared as low‐echogenicity dense images that interrupted the flat, hyperechoic band corresponding to ears’ cartilage (Figure 3). TA‐treated, non‐responsive wounds evolved to larger HTS compared with those derived from GHRP6‐treated, non‐responsive wounds and vehicle‐treated wounds (Figure 4).

Figure 3.

Figure 3

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Sub‐epidermal sonography of the lesions on rabbit's ears. The green segments represent lesion width and depth in centimetres. The hyperechoic band with slightly irregular contours corresponding to ears’ cartilage is interrupted by a low echogenicity area representing the fibrotic core of the hypertrophic scar. The arrow indicates that the cartilage is fully disrupted and displaced by the fibrotic mass corresponding to the hypertrophic scars (HTS). Lesion width and depth was used to estimate HTS size in cm2

Figure 4.

Figure 4

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Prevention experimental scheme. Hypertrophic scar (HTS) size of non‐responsive wounds, estimated by ultrasonographic procedures. Kruskal‐Wallis test followed by Dunn's multiple comparison test. *P < .05, NS, no significance, TA, triamcinolone acetonide

3.1.3. Histopathological findings

The histological analysis (Figure 5) confirmed the macroscopic aesthetic results observed in Figure 2. The qualitative microscopic examination indicated that the primary effect of GHRP6 intervention is translated into a large reduction of the local cellularity and the amount of ECM accumulated. GHRP6‐ and TA‐treated wounds showed SEI mean values (1.1 ± 0.11 and 1.1 ± 0.13, respectively) that were significantly lower than those observed for vehicle‐treated wounds (1.7 ± 0.17; P < .05).

Figure 5.

Figure 5

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Prevention experimental scheme. Histological appearance of representative vehicle‐ (A), GHRP6‐ (B), and triamcinolone acetonide (C)‐treated lesions. Neo‐dermis depth is represented by a black arrow arising from the perichondrium (represented by a black line). Dotted arrows represent normal dermis depth in surrounding intact skin. Yellow line represents the limit of normal neo‐dermis depth as referred to dotted arrows. The segment of the black arrow beyond the yellow line corresponds to exuberant neo‐dermis as a result of matrix accumulation. Haematoxylin/eosin (H/E) ×20 magnification (upper panel) and ×40 magnification (lower panel). This type of images was used to estimate the scar elevation index (SEI). Scale bar corresponds to 200 μm

3.2. Reversion experimental scheme

3.2.1. Macroscopic findings

During the treatment progress, we found no macroscopic differences in consistency, colour, and raised appearance of the GHRP6‐treated scars compared with their saline‐infiltrated counterparts (not shown).

3.2.2. Ultrasonographic characterisation of HTS

In accordance with the macroscopic findings, ultrasonographic data following infiltration 20 indicated no statistical differences in HTS size between the experimental groups (0.194 ± 0.09 vs 0.162 ± 0.071; vehicle vs GHRP6, respectively; P = .0884).

3.2.3. Histopathological findings

On histopathological analysis of the scars, it was surprising to detect, in both GHRP6‐ and vehicle‐treated lesions, the presence of mature bone tissue showing Haversian canals and even a well‐defined marrow (Figure 6).

Figure 6.

Figure 6

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Reversion experimental scheme. Representative images of well‐defined bone tissue (arrow) found within vehicle‐ (A) and GHRP6‐ (B) infiltrated hypertrophic scars. H/E, ×100 magnification. Scale bar corresponds to 200 μm

3.3. Proteomic study

As a result of the proteomics analysis, 687 proteins were identified and quantified, 48 of them with a statistically significant fold change. GHRP6's topical application down‐regulated 23 proteins and up‐regulated 25. A total of 75% of the identified proteins (516) were initially linked to uncharacterised proteins, which were later related to a function regarding their orthologues in humans. The GO analysis showed that 1 hour after exposure to GHRP6, the peptide had triggered significant changes on 9 proteins involved in relevant biological processes in wound healing (Table 1).

Table 1.

Proteins modulated by GHRP6

Variation Accession H/L Homologue entry Gene name Protein name Relevant biological processes
Up‐regulated G1SRL5 2.595 P23141 CES1 Carboxylesterase 1

  • Cholesterol ester hydrolysis involved in cholesterol transport
G1U8B3 2.606 P09455 RBP1 Retinol‐binding protein 1

  • Retinoid metabolic process

  • Vitamin A metabolic process

  • Retinoic acid biosynthetic process
G1T4W4 21.61 Q9UBX5 FBLN5 Fibulin‐5

  • Cell‐matrix adhesion

  • Elastic fibre assembly

  • Extracellular matrix organisation
Down‐regulated G1SD01 0.1425 P11277 SPTB Spectrin

  • Actin filament binding

  • Structural constituent of cytoskeleton
G1TG85 0.1883 P16157 ANK1 Ankyrin‐1

  • Cytoskeleton organisation
G1SRE6 0.2242 P11171 EPB41 Protein 4.1

  • Actin cytoskeleton organisation
G1T676 0.3841 P28289 TMOD1 Tropomodulin‐1

  • Actin filament organisation

  • Pointed‐end actin filament capping
G1TEZ6 0.3068 P07476 IVL Involucrin

  • Keratinocyte differentiation
G1ST78 0.000035 P14543 NID1 Nidogen‐1

  • Extracellular matrix organisation

  • Basement membrane organisation

  • Cell‐matrix adhesion
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Proteins with potential roles in keloids’ pathophysiology that were found to be significantly modulated by GHRP6 1‐hour post‐wounding/treatment are displayed. The histogram with heavy/light (H/L) peptide ratios was tested for normal distribution. H/L ratios higher than 2.19 or lower than 0.45 were considered differentially modulated by GHRP6 (P < .1).

4. DISCUSSION

Regardless of the limitations of this study, GHRP6 intervention has been compellingly shown to prevent the abnormal accumulation of ECM in animals otherwise exhibiting disfigured scars. According to these findings, GHRP6 treatment aborted the onset of HTS in a comparable manner to triamcinolone. Moreover, this desirable effect appeared devoid of local untoward reactions as induced by the first‐line treatment TA. Although TA was topically administered in a reduced dose (2 mg/mL) and in controlled time points in order to minimise wound‐healing arrest, wound stagnation, ulceration and cutaneous atrophy, or reepithelialisation disruption were observed. All of these undesirable side effects, and others linked to TA, have been previously described for corticosteroids in HTS treatment.15 Corticosteroids, and therefore TA, possess numerous pharmacological effects, namely, anti‐inflammatory, antimitotic, apoptotic, vasoconstrictive, and immunomodulatory. All of these properties support these compounds’ efficacy in skin disease treatment16; however, their clinical use is often considered a double‐edged sword as they are frequently accompanied by ‘hard to get rid of’ undesired adverse reactions.15 Although the threads underlying corticosteroids action towards the “good” or the “bad” outcome in clinical practice remain elusive, corticosteroids are still a first‐line therapeutic option for hypertrophic scarring because of the lack of more suitable alternatives with marked effectiveness and less adverse events. This scenario enlightens the potential success of GHRP6 as one of the incoming alternatives for HTS treatment that could overcome corticosteroid‐associated side effects.

According to the histological observations, GHRP6 apparently inhibited perichondrium cells proliferation and reprogramming, which we believe resulted in the impaired cartilage reconstitution and less ECM accumulation observed in the scars treated with GHRP6. This reasoning relies on the assumptions that first: chondrocytes actively contributed to the wound‐healing process by reprogramming themselves into fibroblast‐like cells, and second: that GHRP6 prevented cartilage reconstitution and hypertrophic scarring by directly hindering perichondrium reconstitution. The chondrogenic potential of perichondrium is broadly accepted17, 18; however, the role of perichondrium in scarring is less evident. What is well established is that HTS originate as a result of deep dermis injury19, and the deep dermis and perichondrium share similar tissue architecture as both are considered to be classic examples of dense irregular connective tissue.20 Thus, it is tenable to speculate that both should exert similar responses to trauma. Actually, the outer perichondrium membrane has been pointed to already as being responsible for the fibrous overgrowth seen in wound explants from rabbit ears.21

The microscopic findings discussed above suggest that GHRP6 is somehow attenuating mesenchymal cells’ reactivity in response to injury. In other words, GHRP6 could be hypothetically inhibiting the mesenchyme‐to‐mesenchyme reprogramming process and thus decreasing the surge of fibroblast and myofibroblasts. This notion is supported by previous studies from our group in which GHRP6 was shown to prevent hepatic stellate cell activation and reduce α‐SMA and vimentin expression within the “challenged” liver non‐parenchymal cells. In line with this, GHRP6 also significantly reduced TGF‐β and CTGF expression in both parenchymal and non‐parenchymal cells.8

Even when both GHRP6 and TA significantly aborted the onset of HTS, treatment‐refractory wounds were observed in both medicated groups. The HTS derived from these non‐responsive wounds were larger in the TA‐treated group, which could be related to the stagnant re‐epithelialisation process underwent by these wounds.4 Interestingly, non‐responsive wounds were often located on the same ear and even in the vicinity of responsive wounds that healed without evidences of fibrosis or hyperchromasia. Although intriguing, this does not appear to be a surprising observation in wound‐healing experiments as local fibroblasts may delineate a cutaneous heterogeneity in terms of topographic differentiation and positional/epigenetic memory.22

The reversion scheme implemented here failed to attest any benefit on mature consolidated HTS. Thus, it is reasonable to infer that GHRP6 is not able to reverse this aberrant pathological phenotype in which, among multiple pathogenic factors, a long‐dated epigenetic signature is imprinted.23 In line with this, it is worth mentioning that the histological analysis of the HTS infiltrated along the reversion scheme revealed the presence of well‐defined, bone material, apparently arising as a consequence of a cartilage and/or a fibroblastic ossifying metaplasia.24 This process of heterotopic ossification was not attributable to GHRP6 treatment as it was also detected in the vehicle‐treated HTS. This microscopic observation leads us to believe that fibroblast proliferative niches may subsequently re‐programme to chondroblast‐like cells that eventually differentiate to mature bone tissue with a cellularised marrow and blood vessels.24 This unexpected event may suggest that the evolution time of the HTS until GHRP6 intervention was too long for a proper assessment of the potential success of this peptide in reversing established fibrotic tissue.

The proteomic study conducted here was intended to shed light on the molecular impact of GHRP6 during the very early phases of the healing process. Even though the identification of a greater amount of proteins from these samples was impaired by the presence of highly abundant proteins like keratins, we identified 48 proteins that were differentially expressed in GHRP6‐treated wounds. Nine of them could eventually be associated with the GHRP6 preventing effect as they are involved in relevant biological processes related to lipid metabolism, cytoskeleton arrangements, epidermal cells’ differentiation, and ECM‐cell interaction. The potential role of these 9 proteins in HTS prevention is discussed below.

Carboxylesterase 1 (CES1) and cellular retinol‐binding protein 1 (CRBP1) participate in lipid metabolism.25, 26 As previously noted, GHRP6 is a high‐affinity ligand for CD36,27 a fatty acid translocase implicated in lipid metabolism.28 CES1 up‐regulation is thought to contribute to cholesterol elimination,29, 30 which is ultimately expected to result in reduced inflammation.31 On the other hand, the up‐regulation of CRBP1 could be extrapolated into increased availability of retinoic acid at the injured zone. Retinoids are potential immunosuppressive agents32 that are known to inhibit TGFβ1‐induced collagen expression and impair fibroblast proliferation.33 Taken together, this evidence suggests that the anti‐fibrotic preventing effect of GHRP6 could be partially relying on a possible CES1‐ and CRBP1‐mediated inflammation quenching.

The structural proteins spectrin, protein 4.1, tropomodulin‐1, and ankyrin are components of the “spectrin‐based membrane skeleton.”34 This superstructure is known to influence cell shape and stability.35 Down‐regulation of these proteins as induced by GHRP6 could hinder fibroblasts adhesion and migration,36, 37, 38 thus impeding spreading and survival.

Increased epidermal thickness based on abnormal epidermal differentiation and stratum corneum disorganisation is considered a typical feature of hypertrophic scarring.39 The early differentiation marker involucrin was considered responsible for this phenomenon as it is highly expressed in keloids and HTS.39 Interestingly, GHRP6 significantly down‐regulated involucrin as fast as 1‐hour post‐wounding, which may conceptually suggest that GHRP6 promotes a proper epidermal differentiation.

Fibulin‐5 is involved in proper elastic fibre formation and assembly40 and appears to be insufficiently deposited in keloids’ ECM.41 Additionally, fibulin‐5 has been shown to significantly decrease keloid‐derived fibroblasts proliferation and adhesion rates in vitro.42 Promisingly, GHRP6 was noted to up‐regulate fibulin‐5 expression, which may entail a more physiological elastic fibre deposition, an assembly that could circumvent aberrant scarring. In contrast to fibulin‐5, nidogen‐1 or entactin is highly expressed in pathological fibrotic conditions.43 GHRP6 significantly lowered nidogen‐1expression, thus reducing it to a less fibrogenic state. It is worth pointing out that both fibulin‐5 and nidogen‐1 demonstrated extremely significant fold changes, which may indicate that GHRP6‐mediated early mechanistic response after wounding could be strongly based on ECM composition and dynamics.

All these proteome data propose that GHRP6 may override the fibrogenic process by interfering with multiple pathogenic links involving alternative mechanisms. These “hypothetical mechanisms” are summarised in Figure 7. Nevertheless, a time‐point kinetic study encompassing up to the late remodelling phase and validation methods are required to comprehensively depict the biological underlying the GHRP6‐mediated fibrotic prevention.

Figure 7.

Figure 7

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Biological arms of GHRP6 impact on aesthetic wound healing. GHRP6 was proven to modify the expression pattern of proteins whose biological functions can be related to lipid metabolism, cytoskeleton arrangements, extracellular matrix (ECM)‐cell interaction, and epidermal cell differentiation. As these data derive from an acute and single intervention during an early healing phase, the actual impact of these events in counteracting pathological fibroplasia is hypothetical and supported by the state of the art

Regardless of the evolutional gap existing between rabbits and human beings, which constitutes the main limitation of this work, the results presented here anticipate a potential therapeutic use of GHRP6 in exuberant scar prevention. This peptide has been previously used in clinical practice in the scope of different scenarios, where it was shown to be well tolerated with only minor adverse events.44, 45, 46, 47, 48 In particular, the 400 μg/mL dose evaluated in the present work has been previously assessed intravenously in healthy volunteers and provoked only transient side effects of spontaneous clearance.44 However, GHRP6's possible application in the scenario of fibrosis treatment in humans has never been explored before. Hence, key aspects such as doses, therapeutic schemes, and possible side effects still have to be determined.

In summary, this study enriches the line of evidence documenting a GHRP6‐mediated anti‐fibrogenic effect. Even though its pharmacological indication could be restricted to the prophylactic realm for individuals with exuberant healing history or, simply, to achieve more aesthetic scars, its clinical impact could revolutionise the therapeutic armamentarium for disfiguring wounds. Further studies are fully justified to thoroughly define the potential merits of this unprecedented pharmacological option for cutaneous fibrotic conditions.

Fernández‐Mayola M, Betancourt L, Molina‐Kautzman A, et al. Growth hormone‐releasing peptide 6 prevents cutaneous hypertrophic scarring: early mechanistic data from a proteome study. Int Wound J. 2018;15:538–546. 10.1111/iwj.12895

Present address Lázaro Betancourt, Department of Clinical Sciences, Lund University, Boxes 117, 221 00 Lund, Sweden

Alicia Molina‐Kautzman, Division of Allergy Immunology, University of Sherbrooke, Local E51283 Sherbrooke (Québec) J1K 2R1, Canada

The work should be attributed to the Center for Genetic Engineering and Biotechnology, Department of Pharmaceutics, Wound Healing and Cytoprotection Group.

REFERENCES

  • 1. Seo BF, Jung SN. The immunomodulatory effects of mesenchymal stem cells in prevention or treatment of excessive scars. Stem Cells Int. 2016;2016:1‐8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Zhu Z, Ding J, Tredget EE. The molecular basis of hypertrophic scars. Burns Trauma. 2016;4(2). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Mari W, Alsabri SG, Tabal N, Younes S, Sherif A, Simman R. Novel insights on understanding of keloid scar: article review. J Am Coll Clin Wound Spec. 2016;7(1–3):1‐7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Atiyeh BS. Nonsurgical management of hypertrophic scars: evidence‐based therapies, standard practices, and emerging methods. Aesthetic Plast Surg. 2007;31(5):468‐492. [DOI] [PubMed] [Google Scholar]
  • 5. Berlanga J, Cibrian D, Guevara L, et al. Growth‐hormone‐releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction. Clin Sci (Lond). 2007;112(4):241‐250. [DOI] [PubMed] [Google Scholar]
  • 6. Delgado‐Rubin de Celix A, Chowen JA, Argente J, Frago LM. Growth hormone releasing peptide‐6 acts as a survival factor in glutamate‐induced excitotoxicity. J Neurochem. 2006;99(3):839‐849. [DOI] [PubMed] [Google Scholar]
  • 7. Shen YT, Lynch JJ, Hargreaves RJ, Gould RJ. A growth hormone secretagogue prevents ischemic‐induced mortality independently of the growth hormone pathway in dogs with chronic dilated cardiomyopathy. J Pharmacol Exp Ther. 2003;306(2):815‐820. [DOI] [PubMed] [Google Scholar]
  • 8. Berlanga J, Vázquez D, Cibrián D, et al. Growth hormone releasing peptide 6 (GHRP6) reduces liver fibrosis in CCl4 chronically intoxicated rats. Biotecnol Apl. 2012;29(2):60‐72. [Google Scholar]
  • 9. Mendoza Mari Y, Fernandez Mayola M, Aguilera Barreto A, et al. Growth hormone‐releasing peptide 6 enhances the healing process and improves the esthetic outcome of the wounds. Plast Surg Int. 2016;2016. 10.1155/2016/4361702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Morris DE, Wu L, Zhao LL, et al. Acute and chronic animal models for excessive dermal scarring: quantitative studies. Plast Reconstr Surg. 1997;100(3):674‐681. [DOI] [PubMed] [Google Scholar]
  • 11. Sari E, Bakar B, Dincel GC, Budak Yildiran FA. Effects of DMSO on a rabbit ear hypertrophic scar model: a controlled randomized experimental study. J Plast Reconstr Aesthet Surg. 2017;70(4):509‐517. [DOI] [PubMed] [Google Scholar]
  • 12. O'Shaughnessy KD, De La Garza M, Roy NK, Mustoe TA. Homeostasis of the epidermal barrier layer: a theory of how occlusion reduces hypertrophic scarring. Wound Repair Regen. 2009;17(5):700‐708. [DOI] [PubMed] [Google Scholar]
  • 13. Sanchez A, Gonzalez LJ, Betancourt L, et al. Selective isolation of multiple positively charged peptides for 2‐DE‐free quantitative proteomics. Proteomics. 2006;6(16):4444‐4455. [DOI] [PubMed] [Google Scholar]
  • 14. Yi EC, Marelli M, Lee H, et al. Approaching complete peroxisome characterization by gas‐phase fractionation. Electrophoresis. 2002;23(18):3205‐3216. [DOI] [PubMed] [Google Scholar]
  • 15. Trisliana Perdanasari A, Lazzeri D, Su W, et al. Recent developments in the use of intralesional injections keloid treatment. Arch Plast Surg. 2014;41(6):620‐629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Uva L, Miguel D, Pinheiro C, et al. Mechanisms of action of topical corticosteroids in psoriasis. Int J Endocrinol. 2012;2012:1‐16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Togo T, Utani A, Naitoh M, et al. Identification of cartilage progenitor cells in the adult ear perichondrium: utilization for cartilage reconstruction. Lab Invest. 2006;86(5):445‐457. [DOI] [PubMed] [Google Scholar]
  • 18. Van Osch GJ, Van Der Veen SW, Burger EH, Verwoerd‐Verhoef HL. Chondrogenic potential of in vitro multiplied rabbit perichondrium cells cultured in alginate beads in defined medium. Tissue Eng. 2000;6(4):321‐330. [DOI] [PubMed] [Google Scholar]
  • 19. Wang J, Dodd C, Shankowsky HA, Scott PG, Tredget EE, Wound Healing Research G. Deep dermal fibroblasts contribute to hypertrophic scarring. Lab Invest. 2008;88(12):1278‐1290. [DOI] [PubMed] [Google Scholar]
  • 20. Henrikson RC, Kaye GI, Mazurkiewicz JE. Histology. Baltimore, MD: Williams & Wilkins; 1997. [Google Scholar]
  • 21. Duynstee ML, Verwoerd‐Verhoef HL, Verwoerd CD, Van Osch GJ. The dual role of perichondrium in cartilage wound healing. Plast Reconstr Surg. 2002;110(4):1073‐1079. [DOI] [PubMed] [Google Scholar]
  • 22. Rinn JL, Bondre C, Gladstone HB, Brown PO, Chang HY. Anatomic demarcation by positional variation in fibroblast gene expression programs. PLoS Genet. 2006;2(7):e119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. He Y, Deng Z, Alghamdi M, Lu L, Fear MW, He L. From genetics to epigenetics: new insights into keloid scarring. Cell Prolif. 2017;50(2):e12326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Vanden Bossche L, Vanderstraeten G. Heterotopic ossification: a review. J Rehabil Med. 2005;37(3):129‐136. [DOI] [PubMed] [Google Scholar]
  • 25. Xu J, Yin L, Xu Y, et al. Hepatic carboxylesterase 1 is induced by glucose and regulates postprandial glucose levels. PLoS One. 2014;9(10):e109663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Napoli JL. Cellular retinoid binding‐proteins, CRBP, CRABP, FABP5: effects on retinoid metabolism, function and related diseases. Pharmacol Ther. 2017;173:19‐33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Demers A, McNicoll N, Febbraio M, et al. Identification of the growth hormone‐releasing peptide binding site in CD36: a photoaffinity cross‐linking study. Biochem J. 2004;382(pt 2):417‐424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Silverstein RL, Li W, Park YM, Rahaman SO. Mechanisms of cell signaling by the scavenger receptor CD36: implications in atherosclerosis and thrombosis. Trans Am Clin Climatol Assoc. 2010;121:206‐220. [PMC free article] [PubMed] [Google Scholar]
  • 29. Chistiakov DA, Bobryshev YV, Orekhov AN. Macrophage‐mediated cholesterol handling in atherosclerosis. J Cell Mol Med. 2016;20(1):17‐28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Ghosh S, Zhao B, Bie J, Song J. Macrophage cholesteryl ester mobilization and atherosclerosis. Vascul Pharmacol. 2010;52(1–2):1‐10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Grebe A, Latz E. Cholesterol crystals and inflammation. Curr Rheumatol Rep. 2013;15(3):313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Brown CC, Noelle RJ. Seeing through the dark: new insights into the immune regulatory functions of vitamin A. Eur J Immunol. 2015;45(5):1287‐1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Viera MH, Amini S, Valins W, Berman B. Innovative therapies in the treatment of keloids and hypertrophic scars. J Clin Aesthet Dermatol. 2010;3(5):20‐26. [PMC free article] [PubMed] [Google Scholar]
  • 34. Bennett V. Spectrin‐based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev. 1990;70(4):1029‐1065. [DOI] [PubMed] [Google Scholar]
  • 35. Yamashiro S, Gokhin DS, Kimura S, Nowak RB, Fowler VM. Tropomodulins: pointed‐end capping proteins that regulate actin filament architecture in diverse cell types. Cytoskeleton (Hoboken, NJ). 2012;69(6):337‐370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Bourguignon LY, Zhu H, Shao L, Chen YW. Ankyrin‐Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration. J Cell Biol. 2000;150(1):177‐191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Jung Y, McCarty JH. Band 4.1 proteins regulate integrin‐dependent cell spreading. Biochem Biophys Res Commun. 2012;426(4):578‐584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Sung KL, Yang L, Whittemore DE, et al. The differential adhesion forces of anterior cruciate and medial collateral ligament fibroblasts: effects of tropomodulin, talin, vinculin, and alpha‐actinin. Proc Natl Acad Sci USA. 1996;93(17):9182‐9187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Limandjaja GC, van den Broek LJ, Waaijman T, et al. Increased epidermal thickness and abnormal epidermal differentiation in keloid scars. Br J Dermatol. 2017;176(1):116‐126. [DOI] [PubMed] [Google Scholar]
  • 40. Yanagisawa H, Schluterman MK, Brekken RA. Fibulin‐5, an integrin‐binding matricellular protein: its function in development and disease. J Cell Commun Signal. 2009;3(3–4):337‐347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Ikeda M, Naitoh M, Kubota H, et al. Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid. Biochem Biophys Res Commun. 2009;390(4):1221‐1228. [DOI] [PubMed] [Google Scholar]
  • 42. Furie N, Shteynberg D, Elkhatib R, et al. Fibulin‐5 regulates keloid‐derived fibroblast‐like cells through integrin beta‐1. Int J Cosmet Sci. 2016;38(1):35‐40. [DOI] [PubMed] [Google Scholar]
  • 43. Lai KK, Shang S, Lohia N, et al. Extracellular matrix dynamics in hepatocarcinogenesis: a comparative proteomics study of PDGFC transgenic and Pten null mouse models. PLoS Genet. 2011;7(6):e1002147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Selman‐Housein Bernal K, Hernández Bernal F, Abreu Cruz A, Valenzuela Silva C, Berlanga Acosta J, López SP. Clinical safety of growth hormone‐releasing peptide 6 (GHRP‐6) in healthy volunteers. Invest Medicoquir. 2014;6(1):81‐91. [Google Scholar]
  • 45. Martins MR, Pinto AC, Brunner E, Silva MR, Lengyel AM. GH‐releasing peptide (GHRP‐6)‐induced ACTH release in patients with Addison's disease: effect of glucocorticoid withdrawal. J Endocrinol Invest. 2003;26(2):143‐147. [DOI] [PubMed] [Google Scholar]
  • 46. Oliveira JH, Vieira JG, Abucham J, Lengyel AM. GHRP‐6 is able to stimulate cortisol and ACTH release in patients with Cushing's disease: comparison with DDAVP. J Endocrinol Invest. 2003;26(3):230‐235. [DOI] [PubMed] [Google Scholar]
  • 47. Alaioubi B, Mann K, Petersenn S. Diagnosis of growth hormone deficiency in adults: provocative testing with GHRP6 in comparison to the insulin tolerance test. Horm Metab Res. 2009;41(3):238‐243. [DOI] [PubMed] [Google Scholar]
  • 48. Alaioubi B, Mann K, Petersenn S. Diagnosis of adrenal insufficiency using the GHRP‐6 test: comparison with the insulin tolerance test in patients with hypothalamic‐pituitary‐adrenal disease. Horm Metab Res. 2010;42(3):198‐203. [DOI] [PubMed] [Google Scholar]

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