REPORT
GHRP-6, a novel candidate for prevention and treatment of fibrotic disorders
GHRP-6, un candidato novedoso para la prevención y tratamiento de desórdenes fibróticos
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Yssel Mendoza-Marí, Maday Fernández-Mayola, Dania Vázquez-Blomquist, Ariana García-Ojalvo, José Suárez-Alba, Gerardo Guillén-Nieto, Ana Aguilera-Barreto, Yilian Bermúdez-Álvarez, Dayana Ugarte, Diana García del Barco-Herrera, Ernesto López-Mola, Manuel Selman-Hussein, Jorge Berlanga-Acosta
Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 entre 158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba.
ABSTRACT
Fibrosis is defined as the pathological accumulation of extracellular matrix proteins (ECM) during the tissue repair response to an injury, which interferes with the functioning of the damaged organ or tissue. So far, there are no effective preventive or curative treatments. The growth hormone-releasing peptide 6 (GHRP-6) has anti-inflammatory, anti-oxidant and cytoprotective properties. Early signs of its possible anti-fibrotic effect were observed in a model of dilated cardiomyopathy in rats. This new property of the peptide was first studied in a model of liver cirrhosis in rats, in preventive and therapeutic scenarios. GHRP-6 reduced fibrotic induration in more than 75%, cords thickness and number of cirrhotic nodules by up to 60%, exerting besides a marked hepatoprotective effect. To assess its effect on the skin, GHRP-6 was applied in a simple wound model in rats, where it increased the rate of wound closure and decreased the inflammatory infiltrate. Subsequently, in a model of hypertrophic scarring in rabbits, the peptide prevented the appearance of keloids in more than 90% of the treated wounds. From the molecular point of view, GHRP-6 decreased the transcriptional expression of the pro-fibrotic genes TGFB1 and CTGF and induced the expression of the PPARG and MMP-13 genes, relevant for the inhibition of the pathological cumulative process. This work received the Annual Prize of the Cuban Academy of Sciences for the year 2016.
Keywords: fibrosis, hypertrophic scarring, keloid, GHRP-6, TGFB1, PPARG, wound healing.
RESUMEN
La fibrosis se define como la acumulación patológica de proteínas de la matriz extracelular (MEC) durante la respuesta reparativa tisular frente a una lesión, que interfiere con el funcionamiento del órgano o tejido dañado. Hasta el momento no existen tratamientos preventivos o curativos del todo eficaces. El péptido liberador de la hormona de crecimiento tipo 6 (GHRP-6) posee propiedades anti-inflamatorias, anti-oxidantes y citoprotectoras. Los primeros indicios de su posible efecto anti-fibrótico se observaron en un modelo en ratas de miocardiopatía dilatada. Esta nueva propiedad del péptido se estudió en un modelo de cirrosis hepática en ratas, en escenarios preventivo y terapéutico. El GHRP-6 redujo la induración fibrótica en más del 75%, el grosor de los cordones y el número de nódulos cirróticos hasta en un 60%, además de ejercer un marcado efecto hepatoprotector. Para evaluar su efecto en la piel, el GHRP-6 se aplicó en un modelo de herida simple en rata, donde incrementó la velocidad del cierre de las heridas y disminuyó el infiltrado inflamatorio. Posteriormente, en un modelo de cicatrización hipertrófica en conejos, el péptido previno la aparición de queloides en más del 90% de las heridas tratadas. Desde el punto de vista molecular, el GHRP-6 disminuyó la expresión transcripcional de los genes pro-fibróticos TGFB1 y CTGF e indujo la expresión de los genes PPARG y de MMP-13, relevantes para la inhibición del proceso acumulativo patológico. Este trabajo mereció el Premio Anual de la Academia de Ciencias de Cuba para el año 2016.
Palabras clave: fibrosis, cicatriz hipertrófica, queloide, GHRP-6, TGFB1, PPARG, cicatrización
INTRODUCTION
Fibrosis is defined as the pathological accumulation of extracellular matrix proteins (ECM) during tissue the repair response to mechanical, chemical or biological damage and interferes with the functioning of the affected organ or tissue [1]. Hepatic cirrhosis, cystic fibrosis and keloids are among the diseases displaying this type of process, which show a high incidence rate worldwide, particularly in developed countries, and with no efficacious preventive or curative treatments available [2].
A fortuitous finding made by our research group provided the first observations that the growth hormone-releasing peptide 6 (GHRP-6) establishes a cellular program of degradation or removal of excess ECM in parenchymal organs. GHRP-6 is a six-amino acids synthetic peptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, which was originally obtained as a synthetic derivative of intestinal metaencephalin [3]. At present, this peptide and some of its synthetic analogues are used as growth hormone secretagogues for the clinical diagnosis of different forms of dwarf-ism [4], and they have shown anti-inflammatory, anti-oxidant and cytoprotective properties with good safety profiles [5].
]]> In previous experiments, rats receiving repeated doses of doxorubicin for the induction of dilated car-diomyopathy, developed an intense interstitial fibrosis in the liver, kidneys and lungs. Subsequent his-tological examination showed a substantial decrease in the accumulation of collagen in animals treated with GHRP-6 versus those receiving a placebo solution (unpublished observations). To rigorously study these merely circumstantial findings, the anti-fibrotic effect of the peptide was characterized in several experimental models. First, the molecule was evaluated in a model of liver cirrhosis in rats mediated by carbon tetrachloride intoxication, in a preventive and two therapeutic scenarios. Subsequently, topical application of a viscous composition containing GHRP-6 was assessed in a simple wound model in rats and, afterwards, in a model of hypertrophic scarring in rabbit ears. The results obtained in these experiments will be briefly described below.
RESULTS
Evaluation of the anti-fibrotic effect of GHRP-6 in a model of liver cirrhosis mediated by carbon tetrachloride intoxication in rats
Liver fibrosis was induced by the subcutaneous injection of CCl4 twice a week for five months. The potential effect of the GHRP-6 intervention toward both fibrosis prevention and regression, was examined in two experimental blocks. The first one, developed during the five initial months, included the concomitant administration of GHRP-6 with CCl4 to assess hepatic fibrosis prevention. In the second block of experiments, hepatic fibrosis was established during the first five months and then animals were divided into two groups of treatments: one received GHRP-6 alone for 15 days and the other received GHRP-6 and CCl4 for 60 days. In these two schemes were carried out to assess the peptide potential in promoting cirrhosis regression.
The percentage of fibrosis, nodularity and septal thickness were quantified by histomorphometric studies and ascites or portal dilation by ultrasound. The biochemical profile and the parameters of oxidative stress in serum, as well as the genes and proteins expression pattern, were determined by reverse transcription associated to polymerase chain reaction and by immunohistochemistry.
Treatment with GHRP-6 significantly reduced fibrosis markers such as fibrosis index, portal diameter, presence of ascites, thickness of the walls and number of nodules per field, both from the ultrasonographic and histological point of view (Table 1 and Table 2, respectively), in both concomitant and therapeutic approaches. Representative images of the macroscopic and histological structure of livers before and after GHRP-6 peptide administration are shown in Figure 1.
From the functional point of view, it was observed that the peptide significantly attenuated the dramatic increase in the levels of the transaminases aspartate-aminotransferase and alanine-aminotransferase, which occurred in the animals receiving the hepatotoxic agent. On the other hand, GHRP-6 significantly reduced the levels of oxidative stress markers as compared to saline-treated animals (p < 0.01) in the three experimental schemes, including the levels of total hydroperoxides, advanced protein oxidation products, malondialdehyde and the lipid peroxidation potential. Concurrently, GHRP-6 increased the activity of the anti-oxidant enzymes catalase and superoxide dismutase (p < 0.05). In general, we could conclude that the GHRP-6 intervention removed and controlled the pathological deposition of collagen and ECM in the hepatic parenchyma, while exerting marked hepatoprotective and proliferation promoter effects [7].
Topical application of a carboxymethylcellulose gel containing GHRP-6 in the simple open excision wound model in rats
]]> More than a decade ago, CD36 was identified as one of the GHRP-6 receptors [8]. Serendipitous observations of our laboratory indicated that CD36 mRNA transcript appeared abundantly represented in clinical samples of granulation tissue of either acute (deep burn injuries) or chronic (pressure ulcers) wounds. This finding incited us to speculate about the possible effect of GHRP-6 topical administration on the wound healing process.A formulation of GHRP-6 (400 μg/mL) in 1 % carboxymethylcellulose gel (CMC) was applied for 4 days in controlled full-thickness skin wounds, which were surgically made on the back of male Wistar rats. As shown in Figure 2, the administration of GHRP-6 significantly increased wound closure rate as compared to vehicle (CMC 1 %), starting 24 h after the initial administration of the peptide (p = 0.016) and until the end of the experiment (p < 0.0001). Wounds treated with GHRP-6 showed lower amounts of inflammatory infiltrate and attained a higher degree of organization of their ECM, due to a lower accumulation of fibrin and the presence of thinner and horizontally distributed collagen strands. No statistically significant differences were observed in the number of active blood vessels (Table 3).
At the molecular level, GHRP-6 reduced the transcriptional expression of genes encoding tumor necrosis factor converting enzyme (Adam17; p = 0.0306), transforming growth factor β1 (Tgfb1; p = 0.0171) and connective tissue growth factor (Ctgf; p = 0.001). These effects were translated into a decreased expression of genes coding for ECM proteins and for myofibroblast marker proteins [9].
Demonstration of the anti-fibrotic effect of GHRP-6 in the keloid model on the inner surface of the rabbit ear
White male New Zealand rabbits (4.3-4.5 kg) were used in four independent and extemporaneous experiments. Three to four wounds were created on the ventral side of each ear, down to the surface of the cartilage, using a 6 mm diameter punch biotome. Rabbits were randomly assigned to either GHRP-6 (400 μg/mL) treatment or 1 % CMC placebo gel. Treatments were initiated immediately after surgery and continued thereafter until day 30 and the animals remained in observation for another 20 days after GHRP-6 administration had been completed. The most notable effect of the GHRP-6 intervention was the prevention of hypertrophic scar formation. As shown in Table 4, treatment with the peptide abrogated the debut of keloids in 90.5 % of the treated wounds. Conversely, 87.5 % of the wounds that received the viscous solution of 1 % CMC without the peptide evolved into a hypertrophic scar, with a nipple-like appearance, reddened and firm in touch (Figure 3).
GHRP-6 appears to primarily reduce the local hypercellularity associated with cartilage perichondrium cells and the resulting accumulation of ECM (Figure 4). Consequently, the scar elevation index was significantly lower (p = 0.001) in treated wounds (1.12 ± 0.11) than those receiving the vehicle (1.62 ± 0.15). At the molecular level, GHRP-6 significantly reduced the transcriptional expression of Tgfb1 and Ctgf (p < 0.05) and increased the expression of the PPARγ transcriptional factor (p = 0.016) [9].
RELEVANCE OF THE STUDY
The results presented above constitute the first evidence of a new pharmacological property for GHRP- 6: its ability to inhibit synthesis and excessive accumulation of extracellular matrix proteins. This effect was demonstrated in parenchymal (liver) and peripheral (skin) organs and in prophylactic and therapeutic scenarios. On the other hand, a new mechanism of action for GHRP-6, based on the induction of PPARγ, is described. Evidence obtained indicates that GHRP-6 is potentially useful for the prevention/treatment of liver fibrosis, keloids and hypertrophic scars and broadens the spectrum of therapeutic possibilities to other fibrotic and accumulation diseases.
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ACKNOWLEDGEMENTS
The authors thank the valuable cooperation of the following collaborators: María Elena Ochagavía Roque, Jamilet Miranda Navarro, Ricardo Bringas Pérez, Karelia Cosme Díaz, Daniel Palenzuela Gardón, Julio Raúl Fernández Massó, Isabel Guillén Pérez, Alberto Cintado Benítez, Lidia Inés Novoa, José Ángel Silva Guirado, Regla Estrada Vázquez, from the Center for Genetic Engineering and Biotechnology, CIGB; Yolanda González Ferrer, José M. Vila, Angel Abreu, Yolanda Cruz, Ivon Howland, Aleida Urquiza, from the from Center for Medical and Surgical Research, CIMEQ, Cuba; Ana Janet Mir Benítez from the Clinical and Surgical Hospital Joaquín Albarrán, Havana, Cuba; Rosa María Coro Antich, from the Neurology and Neurosurgery Institute, INN, Cuba; and Olga Sonia León Fernández, from the Institute of Pharmacy and Food, IFAL, Havana, Cuba.
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Jorge Berlanga-Acosta. Centro de Ingeniería Genética y Biotecnología, CIGB. Ave. 31 entre 158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba. E-mail: jorge.berlanga@cigb.edu.cu.
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