Eleven-amino acid peptides that mimic the erythropoietin α-helix B increases cell survival in endotheliocyte culture

El péptido de once-aminoácidos que mimetiza la α-hélice B de la eritropoyetina aumentan la supervivencia celular en el cultivo de endoteliocitos

Oleg V Antsiferov

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Mikhail V Korokin korokin@bsu.edu.ru

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Vladimir V Gureev

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Mikhail V Pokrovskii

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Liliya V Korokina

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Oleg S Gudyrev

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Tatiana G Pokrovskaia

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Vladislav O Soldatov

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Galina A Batishcheva

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

Aleksandr A Stepchenko

Belgorod State University, 85, Pobedy St., Belgorod, 308015, Russia, Rusia

This work is licensed under Creative Commons Attribution-NoDerivs 4.0 International.

It is now known that vascular endothelium has a wide range of functions, and maintenance of endothelial homeostasis is a multidimensional active process, disruption of which has a potentially deleterious consequence if not reversed¹. Endothelial cells injury leads to the development of endothelial apoptosis and dysfunction, the consequences of which can be local and general. Currently, the role of vascular endothelium in the pathogenesis of acute and chronic inflammatory processes is attracting increasing attention, especially in relation to cardiovascular diseases².

Endothelial cell dysfunction (ECD) manifested in lesion-prone areas of the arterial vasculature, results in the earliest detectable changes in the life history of an atherosclerotic lesion — the focal permeation, trapping, and physicochemical modification of circulating lipoprotein particles in the subendothelial space. Changes in the spectrum of molecules secreted and expressed by endothelium and smooth muscle cells such as chemokines, IL-1, monocyte chemoattractant protein-1, granulocyte-monocyte stimulating factor, and IL-8, disruption of endothelial barrier function, and ultimately leads to infiltration of the vascular wall by atheromatous masses, and the formation of atherosclerotic plaques³. In turn, chronic and acute overproduction of reactive oxygen species (ROS) develops into cardiovascular diseases (CVD) and, in particular, atherosclerosis⁴. The emerging pathogenetic cascade becomes a relevant target for the pharmacological effects of drugs.

In the search for compounds with endothelio-protective activity allows, substances with a pronounced cytoprotective effect play an important role in reducing oxidative stress manifestations, acute and chronic inflammation leading to atherogenesis.

One of these compounds with pronounced universal cytoprotective activity is erythropoietin (EPO). Our previous studies have shown that recombinant erythropoietin and carbamylated EPO (CEPO) have tissue-protective, endothelial, and cardioprotective effects in various experimental models^5,6,7,8. It has been reported that EPO can bind the tissue-protective receptor (TPR, namely EPOR/CD131 heterodimer) and plays an important role in tissue protection and immune regulation. It was suggested that the helix B of EPO, which is exposed to aqueous medium away from the binding sites of EPO and (EPOR)2, is critical for the recognition of TPR, and it was confirmed that the helix B peptide had similar tissue-protective effects for EPO and CEPO. Based on these observations, an eleven-amino acid linear peptide, QEQLERALNSS, mimicking the three-dimensional structure of the external aqueous face of the helix B peptide was developed and named helix B surface peptide (HBSP)⁹.

In the present study, we assessed the cytoprotective effect on the HUVEC cell culture of the following derivatives of the 11-membered peptide HBSP (QEQLERALNSS): EP-11-1 (UEHLERALNSS), EP-11-2 (UEQLERALNCS), EP-11-3 (UEQLERALNTS), peptides that mimic the spatial structure of the erythropoietin helix B in vitro. Amino acid sequences were obtained by homologous analysis to determine groups of related peptides of the parent compound using the BLAST program.

The experimental study was conducted at the Research Institute of Pharmacology of Living Systems of Belgorod State University. The study was performed in compliance with the requirements of General Requirements for the Competence of Testing and Calibration Laboratories 17025-2009, GOST R ISO 5725-2002 and the Rules of Laboratory Practice, approved by Order of the Ministry of Healthcare and Social Development of the Russian Federation dated August 23rd, 2010 № 708n.

The study was performed using the primary culture of human endotheliocyte cells HUVEC (Sigma-Aldrich, Merck). Endothelial cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20 mM HEPES buffer, 5 U/ml heparin, 200 μg/ml E CG F (Sigma-Aldrich), 10% fetal calf serum (FBS) at 37°C in a humid atmosphere containing 5% CO₂^10,17.

Cells were scattered in 96-well gelatin-coated plates with a density of 5 thousand cells per well. After 24 h of incubation, the studied peptides (HBSP, EP-11-01, EP-11-02, EP-11-03) were added in 3 concentrations - 5 μg/ml, 30 μg/ml and 50 μg/ml. Thus, 3 experimental plates were formed, one for each studied peptide concentration.

Table 1
Protocol for the plate wells in the study of the cytoprotective activity of innovative peptides (concentration 5 μg/ml).

Three hours after the addition of the peptides, hydrogen peroxide (H₂O₂) was added to the plate wells at a final concentration of 200 μM. After 24 hours, the culture medium containing H₂O₂ was replaced with normal.

ell viability was measured using the MTT test¹⁹ developed by Mosman (1983), which consists of the formation of formazan purple from a tetrazolium salt, MTT (3- (4,5-dimethyl thiazol-2-yl bromide) -2,5-diphenyl tetrazolium), where this salt is reduced by the action of the mitochondrial enzyme succinate dehydrogenase. Thus, the level of mitochondrial respiration of the cell was assessed by the rate of accumulation of formazan crystals in the cytoplasm, which is an indicator of its viability. The amount of formazan formed in the cell monolayer is proportionally related to the number of living cells in the test sample.

At the end of the experiment, the medium was selected, a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT, Sigma) in PBS (Sigma) containing 0.9 mM CaCl₂ was added to the wells of the plate and 0.5 mM MgCl₂, based on 10 mg MTT per well. After a 4-hour incubation at 37°C in a humid atmosphere containing 5% CO2, the plate was unscrewed at 600 g on a R4810 centrifuge (Eppendorf, Germany). The supernatant was carefully aspirated.

DMSO, 250 μl/well was used to dissolve the formed crystals of formazan. After the complete dissolution of the crystals, 150 μl, without affecting the cell layer on gelatin, was transferred to a new plate. The optical density of a solution of formazan in DMSO was measured on a Multiscan EX spectrophotometer at a wavelength of 570 nm. Assessment of the results of the MTT test was carried out by comparing the optical density in the experimental and control wells. The number of viable cells was calculated by the formula N = AU experimental / AU control * 100% Statistical data processing was performed using the Kruskal-Wallis test followed by Dunn test (ANOVA). Data are presented as mean ± s.d. Data were considered significant at p≤ 0.05.

To model oxidative stress, using hydrogen peroxide (Н₂О₂) (200 μM), stimulates programmed cell death — apoptosis, which is accompanied by several biochemical and morphological changes in the cell: chromatin condensation, DNA fragmentation, increased membrane permeability and cell compression^11,18.

The average optical density in the experimental groups is shown in Table 1.

Our results show that oxidative stress induced by H₂O₂ produces a significant decrease in the optical density of the medium and the percentage of viable cells of the HUVEC (Table 1, figure 1).

Analysis of Table 1 in which are shown the medium optical density values, with a HUVEC cell culture, against the background of oxidative stress and in the presence of the different peptides at three concentrations, show that the studied compounds have a statistically significantly cytoprotective effect, increasing mitochondrial activity and cell survival in cell culture. Compound EP-11-1A showed a dose-dependent increase in the level of mitochondrial activity of cells in the culture. The increase in the level of cytoprotective activity with the innovative peptides EP-11-1 and EP-11-2 were similar to the percentage of viable cells in the HUVEC culture with the addition of hydrogen peroxide, while EP-11-1 at a concentration of 50 μg/ml was statistically significantly higher (p<0.05) than in the experimental series with the base peptide HBSP (Table 1, figure 1).

Evaluation of the results obtained in the analysis of the MTT test in a series of experiments with the EP-11-3 peptide, comparing the density of the formazan solution and the percentage of surviving endotheliocytes between the series of experiments with the HBSP base peptide and its derivative with laboratory code EP-11-3 allows us to conclude that the compound has the highest cytoprotective activity. Despite the absence of a pronounced dose-dependent effect, a statistically significant (p<0.05) increase in cell survival was found over the entire range of studied EP-11-3 concentrations (5, 30, and 50 μg/ml) (Table 1, figure 1).

Figure 1
The number of viable cells in the experimental groups with the addition of innovative peptides at concentrations of 5 30 and 50 μgml *p 005 when compared with HUVEC **p005 when compared with HUVEC H2O2 #005 when compared with HBSP HUVEC H2O2

As part of the possible approaches to the pharmacological treatment of endothelium-associated pathology, we previously studied the endothelio-protective activity of several compounds, drugs, and their combinations on various experimental models of endothelial dysfunction^12,13,14.

In effect, Michael Brines et al. (2004) demonstrated that the non-hematopoietic effects of erythropoietin are mediated through the heterodimeric EPOR/CD131 complex. The finding that the erythropoietic and tissue-protective properties of EPO are mediated through two different receptor systems has led to the creation of prerequisites for a fundamentally new direction in the search for innovative molecules with cytoprotective activity. In 2008, the same authors demonstrated the cytoprotective activity of an 11-amino acid peptide based on the α-helix B of erythropoietin that mimics the spatial part of a molecule that interacts with a heterodimeric EPOR/CD131 receptor but does not interact with a homodimeric EPOR/EPOR receptor. This peptide HBSP demonstrated the ability to significantly improve the morphofunctional state of tissues in diabetic retinal edema, renal ischemia-reperfusion, and significantly improve cognitive functions in the galantamine-induced amnesia model, in the absence of any effect on erythropoiesis⁹.

Our previous results on the study of the endothelioprotective activity of the HBSP base peptide with the amino acid sequence QEQLERALNSS, using the L-NAME treatment to induceendothelial dysfunction, we demonstrated that HBSP has a pronounced endothelial-protective and potentially atheroprotective effect due to its ability to prevent the death of endothelial cells, as well as to reduce remodeling and pro-inflammatory activation of the vascular wall. Nevertheless, the prothrombotic activity of the peptide limits its use as a preventing and treating agent for atherosclerosis-associated diseases¹⁵. Thus it is advisable to search for peptides related to the original HBSP which that does not have prothrombotic activity, or that have anticoagulant properties. The search for such compounds can be solved in several ways, including adding amino acid motifs with anticoagulant properties to the amino acid sequence, or by searching for groups of related peptides of the parent compound using the BLAST program. The peptides analyzed in this study were obtained using the second approach - screening using the BLAST program¹⁶.

At the first stage of the study of the pharmacological activity of the obtained derivatives. They were used to study of the cytoprotectionactivity of HBSP derivatives under laboratory codes EP-11-1 (UEHLERALNSS), EP-11-2 (UEQLERALNCS), EP-11-3 (UEQLERALNTS). Assessment of cell survival in a culture of human endotheliocytes under conditions of oxidative stress in the presence of the studied peptides showed that the original HBSP peptide and its derivatives EP-11-1, EP-11-2, EP-11-3 have a pronounced (p<0.05 in compared with control) cytoprotective action. Derivatives EP-11-1, EP-11-2 do not differ from the base peptide HBSP in terms of the level of detected activity. When comparing the density of the formazan solution and the percentage of surviving endotheliocytes between the series of experiments with the base peptide HBSP and its derivative with the laboratory code EP-11-3, a statistically significant (p <0.05 compared with HBSP) superiority of EP-11-3 was found over the entire range studied concentrations. Thus, we confirmed that our proposed compounds that mimic the erythropoietin B α-helix have pronounced cytoportective activity, and further studies are required to answer the questions of using these compounds as peptides with endothelioprotective and antiplatelet activity.

11-amino acids derivatives of EPO with laboratory codes EP-11-1, EP-11-2 do not differ in the cytoprotective action capacity when compared with the base peptide HBSP. The derivative with laboratory code EP-11-3 presents a higher significantly cytoprotective activity compared to the base peptide HBSP.