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The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage, downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex, and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

To reveal the association of plasma level of high density lipoprotein cholesterol (HDL-C) level with the transcript level of annotated genes in peripheral blood mononuclear cells (PBMC) and involved in HDL metabolism and atherogenesis at the absence of morphologically evident coronary stenosis.Transcript levels of 63 genes in PBMC from 38 male patients 40-60 years without coronary atherosclerosis with widely varied HDL-C level were measured. The protein interactions were analyzed with STRING database.Among 22 HDL-related genes, the transcript levels for 10 genes (Coordinate regulation of cholesterol influx and efflux in PBMC in atherosclerosis-free subjects with widely varied HDL-C level is suggested. The decreased synthesis and transport of cholesteryl ester to the liver may contribute to hyperalphalipoproteinemia. HDL-C increase is associated with the decrease of expression of innate immunity and inflammation genes. Visualization of 22 responder genes is suggested to be useful in the validation of HDL functionality and atherogenesis even at the absence of morphologically evident coronary stenosis.Cilj rada je otkrivanje povezanosti nivoa lipoproteinskog holesterola visoke gustine u plazmi (HDL-C) sa nivoom transkripta anotiranih gena u mononuklearnim ćelijama periferne krvi (PBMC), a koji su uključeni u HDL metabolizam i aterogenezu u odsustvu morfološki evidentne koronarne stenoze.Izmereni su nivoi transkripta 63 gena u PBMC kod 38 pacijenata muškog pola starosti između 40 i 60 godina bez koronarne ateroskleroze sa značajno raznovrsnim nivoom HDL-C. Interakcije proteina su analizirane pomoću STRING baze podataka.Među 22 gena povezana sa HDL, nivoi transkripta za 10 gena (ABCA1, BMP1, CUBN, HDLBP, LCAT, LDLR, PRKACB, PRKACG, SCARB1 i ZDHHC8) su bili u negativnoj korelaciji sa HDL-C, dok je korelacija pozitivna za GEN APOA1. Od 41 gena koji su skloni aterosklerozi, nivoi transkripta za 11 gena (Predlaže se koordinisana regulacija priliva i odliva holesterola u PBMC kod subjekata bez ateroskleroze sa širokim opsegom vrednosti nivoa HDL-C. Smanjena sinteza i transport holesterol estra do jetre može doprineti hiperalfalipoproteinemiji. Povećanje HDL-C povezano je sa smanjenjem ekspresije gena u vezi sa urođenim imunitetom i upalnim procesima. Vizualizacija ovih 22 gena može biti korisna u validaciji HDL funkcionalnosti i aterogeneze, čak i u odsustvu morfološki vidljive koronarne stenoze.

Cerebral ischaemia is the most common cause of impaired brain function. Biologically active peptides represent potential drugs for reducing the damage that occurs after ischaemia. The synthetic melanocortin derivative, ACTH(4-7)PGP (Semax), has been used successfully in the treatment of patients with severe impairment of cerebral blood circulation. However, its molecular mechanisms of action within the brain are not yet fully understood. Previously, we used the transient middle cerebral artery occlusion (tMCAO) model to study the damaging effects of ischaemia&ndash, reperfusion on the brain transcriptome in rats. Here, using RNA-Seq analysis, we investigated the protective properties of the Semax peptide at the transcriptome level under tMCAO conditions. We have identified 394 differentially expressed genes (DEGs) (&gt, 1.5-fold change) in the brains of rats at 24 h after tMCAO treated with Semax relative to saline. Following tMCAO, we found that Semax suppressed the expression of genes related to inflammatory processes and activated the expression of genes related to neurotransmission. In contrast, ischaemia&ndash, reperfusion alone activated the expression of inflammation-related genes and suppressed the expression of neurotransmission-related genes. Therefore, the neuroprotective action of Semax may be associated with a compensation of mRNA expression patterns that are disrupted during ischaemia&ndash, reperfusion conditions.

A search for genes involved in the molecular mechanisms of atherogenesis and the atheroprotective role of HDL has been carried out to study these processes on the transcriptome level. Bioinformatic analysis of gene expression in peripheral blood of patients with coronary artery stenosis using data (GSE20129, GSE12288, GSE10195, GSE20686) from the GEO genome-wide studies database revealed 947 differentially expressed genes. Of these, 66 genes associated with atherosclerotic coronary artery disease, underlying coronary heart disease (CHD) and myocardial infarction, have been selected. We added to this list the genes for which the association with coronary artery atherosclerosis was determined by the authors of abovementioned genome-wide researches using bioinformatic algorithms (19 genes) or RT-PCR (67 genes), as well as 21 genes involved in HDL metabolism according to Reactome database. Analysis of the resulting list of genes revealed that 60 of them were took part in lipoprotein and lipid metabolism, reverse cholesterol transport, and inflammation. We suggest that these genes are involved in HDL metabolism and atherogenesis in CHD patients. Evaluation of their differential expression in human peripheral blood cells can be applied for further study of the molecular mechanisms of coronary artery atherosclerosis and the atheroprotective role of HDL.

Atheroprotective properties of human plasma high-density lipoproteins (HDLs) are determined by their involvement in reverse cholesterol transport (RCT) from the macrophage to the liver. ABCA1, ABCG1, and SR-BI cholesterol transporters are involved in cholesterol efflux from macrophages to lipid-free ApoA-I and HDL as a first RCT step. Molecular determinants of RCT efficiency that may possess diagnostic and therapeutic meaning remain largely unknown. This review summarizes the progress in studying the genomic variants of ABCA1, ABCG1, and SCARB1, and the regulation of their function at transcriptional and post-transcriptional levels in atherosclerosis. Defects in the structure and function of ABCA1, ABCG1, and SR-BI are caused by changes in the gene sequence, such as single nucleotide polymorphism or various mutations. In the transcription initiation of transporter genes, in addition to transcription factors, long noncoding RNA (lncRNA), transcription activators, and repressors are also involved. Furthermore, transcription is substantially influenced by the methylation of gene promoter regions. Post-transcriptional regulation involves microRNAs and lncRNAs, including circular RNAs. The potential biomarkers and targets for atheroprotection, based on molecular mechanisms of expression regulation for three transporter genes, are also discussed in this review.

Brain stroke continues to claim the lives of million people every year. To build the effective strategies for stroke treatment it is necessary to understand the neuroprotective mechanisms that are able to prevent the ischemic injury. Consisting of the ACTH(4-7) fragment and the tripeptide Pro-Gly-Pro (PGP), the synthetic peptide Semax effectively protects brain against ischemic stroke. However, the molecular mechanisms underlying its neuroprotection and participation of PGP in them are still needed to be clarified. To reveal biological processes and signaling pathways, which are affected by Semax and PGP, we performed the transcriptome analysis of cerebral cortex of rats with focal cerebral ischemia treated by these peptides. The genome-wide biochip data analysis detected the differentially expressed genes (DEGs) and bioinformatic web-tool Ingenuity iReport found DEGs associations with several biological processes and signaling pathways. The immune response is the process most markedly affected by the peptide: Semax enhances antigen presentation signaling pathway, intensifies the effect of ischemia on the interferon signaling pathways and affects the processes for synthesizing immunoglobulins. Semax significantly increased expression of the gene encoding the immunoglobulin heavy chain, highly affects on cytokine, stress response and ribosomal protein-encoding genes after occlusion. PGP treatment of rats with ischemia attenuates the immune activity and suppresses neurotransmission in the CNS. We suppose that neuroprotective mechanism of Semax is realized via the neuroimmune crosstalk, and the new properties of PGP were found under ischemia. Our results provided the basis for further proteomic investigations in the field of searching Semax neuroprotection mechanism.

Complexin 2 is a cytosolic protein participating in synaptic-vesicle exocytosis. The RT-PCR technique was used in a real-time study of the dynamics of variation of expression of the Cplx2 gene that encodes this protein. The study was performed in the rat brain. Measurements under the conditions of incomplete global ischemia were performed during 1 day; measurements under the conditions of focal ischemia were carried out 3, 24, and 72 h after irreversible occlusion of the medial cerebral artery. The most significant decrease in Cplx2 transcripts under the conditions of incomplete global ischemia was observed in the hippocampus and the frontal cortex of rats 12 and 24 h after irreversible occlusion of the common carotid arteries, respectively. Under conditions of focal ischemia, a decrease in the content of Cplx2 transcripts was observed only in focal lesions of the frontoparietal cortex 24 h after occlusion. The effect of Semax neuroprotector peptide and its C-terminal fragment Pro-Gly-Pro on Cplx2 expression was examined under the model ischemia conditions. In the case of incomplete global ischemia, application of Semax compensated for the maximum decrease in Cplx2 expression in the frontal cortex and hippocampus in rats caused by lesions. However, the peptide had no effect when used in rats with focal ischemia. The effect of PGP on Cplx2 expression under conditions of experimental ischemia was more complicated and did not always coincide with the effect of Semax. The obtained results provide insight into the specific features of Cplx2 expression under the conditions of experimental brain ischemia and help to better understand the mechanisms of activity of peptide preparations.

Neurotrophins stimulate the regeneration of neural tissue after lesions. It is also known that the sources of neurogenesis and cerebral function recovery are predominantly located in subcortical brain structures. The effects of ischemia on the expression of genes that encode neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion site were studied 3, 24, and 72 h after irreversible unilateral occlusion of the middle cerebral artery in rats. Changes in the mRNA expression of these genes were assessed by relative quantification using real-time RT-PCR. Sham surgery was found to stimulate the expression of genes that encode neurotrophins (Bdnf, Ngf) and their receptor (p75). It has been shown that ischemia influenced the expression of neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion focus, including the contralateral hemisphere. The downregulation of Bdnf and TrkB transcripts and Ngf and TrkA upregulation in the contralateral cortex on the first day of ischemia obviously reflected stress response. On day 3, Nt-3 transcription increased in all investigated structures outside the lesion focus. In the contralateral hemisphere, relative levels of TrkA and TrkC mRNA expression increased, while p75 expression decreased. Presumably, the observed changes in gene transcription serve to facilitate neuroplasticity and neural tissue regeneration.

The heterogeneity and content of human plasma high-density lipoprotein (HDL) related to their atheroprotective properties determined by various molecular and cellular mechanisms still remain to be completely clarified. For 29 atherosclerosis-free male subjects, we studied the relationship of plasma lipid levels and the content of apolipoprotein A-I (apoA-I)-containing HDL with preβ-electrophoretic mobility, the efficiency of BODIPY-cholesterol efflux from RAW 264.7 macrophages to apolipoprotein B (apoB)-deficient plasma, and the expression level of 22 genes related to HDL metabolism in mononuclear cells. A significant decrease in the absolute content of apoA-I in preβ-HDL was found in subjects with hypoalphalipoproteinemia compared with the subjects with hyperalphalipoproteinemia. The preβ-to-α-ratio of the apoA-I content was constant within the HDL-cholesterol (HDL-C) range 0.59 to 2.24 mM. However, this ratio was significantly increased with an increase in the plasma triacylglycerol (TAG) content from 0.59 to 3.42 mM. A correlation of the level of preβ-HDL with the basal and ABCA1-mediated efflux of cholesterol is shown. The transcript levels for six HDL-metabolizing genes (LDLR, LCAT, ABCA1, SCARB1, ZDHHC8, and BMP1) were decreased, while the transcript level of APOA1 gene was increased in mononuclear cells of subjects with hyperalphalipoproteinemia as compared with subjects with hypoalphalipoproteinemia. A reduction of the intracellular cholesterol level and inhibition of the expression of cholesterol transporters by nascent HDL in mononuclear cells from subjects with hyperalphalipoproteinemia are suggested. Hyperalphalipoproteinemia can be a driving force of the decreased flux of cholesteryl ester to the liver and the increased TAG hydrolysis. The atheroprotective effect of preβ-HDL in hypertriglyceridemia is proposed.

The peptide Semax effectively protects brain tissues against ischemic stroke. However, the molecular mechanisms that underlie its action remain unknown. We used the focal cerebral ischemia rat model with permanent middle cerebral artery occlusion (pMCAO). During the experiments, animals were given intraperitoneal injections of Semax, Pro-Gly-Pro, or saline. We studied the effect of the peptides on the expression of more than 80 growth factor genes in the cortex. As a response to Semax administration, alterations in the expression of growth factor genes were detected at 3, 24, and 72 h after pMCAO. The most pronounced effects of Semax, i.e., the downregulation of the transcripts of 20 genes and the upregulation of 12 growth factor genes, were observed 3 days after artery occlusion. According to our data, Semax promoted the upregulation (by ≥10-fold) of the Csf3 and Artn genes, as well as of the cytokine genes Il1b and Il6. The peptide products of these genes have regulatory properties and exert neuroprotective effects in injured brain tissues. We presume that Semax triggers neuroprotective mechanisms by affecting these systems via the regulation of the expression of growth factor genes.

Tripeptide Pro-Gly-Pro (PGP) belongs to the group of biologically active regulatory peptides that are resistant to peptidases. This makes it possible to use it as a C-terminal fragment of peptide agents Semax and Selank. In recent years, the independent effects of PGP were revealed. The question was raised of whether PGP contributes to the action of pharmaceutical agents that contain this peptide as a fragment. Genome-wide analysis was performed to examine the PGP impacts on the transcriptome of rat cerebral cortex in experimental ischemia. We compared the gene expression levels in animals exposed to ischemia and PGP and in rats of the ischemia control group in 3 and 24 h after the permanent occlusion of the left middle cerebral artery. The altered expression of 29 genes was detected in 3 h, while the altered expression of 57 genes was detected in 24 h. The proteins encoded by these genes fulfill various functions, such as cytokines, transport proteins, transcription factors, transmembrane receptors, etc. Biological processes associated with the genes with altered expression under experimental conditions were determined. It was shown that PGP affects a number of processes involved in the functioning of various body systems. The expression of genes associated with the immune response process changed most significantly a day after the occlusion of the middle cerebral artery. The predominant effect of the suppression of the expression of the immune system genes was observed.

Brain stroke continues to claim the lives of million people every year. To build the effective strategies for stroke treatment it is necessary to understand the neuroprotective mechanisms that are able to prevent the ischemic injury. Consisting of the ACTH

Consisting of a fragment of ACTH(4-7) and C-terminal PGP tripeptide, the polypeptide Semax is successfully used for acute stroke therapy. Previous experiments showed rapid induction of Bdnf, Ngf, and TrkB expression in intact rat hippocampus following Semax treatment. To investigate the mRNA expression of neurotrophins and their receptors after treatment with either Semax or PGP, the rat brains were analyzed at three time points following a permanent middle cerebral artery occlusion (pMCAO). We have shown for the first time that both Semax and PGP activate the transcription of neurotrophins and their receptors in the cortex of rats subjected to pMCAO. The profiles of transcription alteration under PGP and Semax treatment were partially overlapped. Semax enhanced the transcription of Bdnf, TrkC, and TrkA 3 h after occlusion, Nt-3 and Ngf 24 h after occlusion, and Ngf 72 h after occlusion. PGP enhanced the transcription of Bdnf and TrkC 3 h after pMCAO and Ngf, TrkB, TrkC, and TrkA 24 h after pMCAO. The analysis of the transcription alterations under PGP and Semax treatment in the cortex of rats without surgery, sham-operated rats and rats subjected to pMCAO revealed that Semax selectively affected the transcription of neurotrophins and their receptors in the ischemic rat cortex, whereas the influence of PGP was mainly unspecific.

Metabolites of the sphingomyelin cycle are reported to play an important role in neuronal death after ischemia. To elucidate the involvement of the key enzyme of this cycle, sphingomyelin synthase (SMS), in the mechanism underlying cerebral ischemia, we, for the first time, investigated changes in the mRNA expression of the SMS1 gene in rats after focal cerebral ischemia. According to our histological analysis, the damaged area is localized only in the ipsilateral cortex. In the ischemic cortex, the level of SMS1 transcripts was decreased at 3 and 24 h after occlusion, and at 72 h it had returned to the control level. A reduced level of SMS1 mRNA expression in the subcortex of rats with occlusion and sham-operated animals also was appeared during the first 24 h after surgery. This could be attributed to the effect of surgical stress. Seventy-two hours after occlusion, SMS1 mRNA expression in subcortex of ischemic rats was still at a decreased level; this may be considered to be a somewhat distant extended effect. Our results show the early response of the SMS1 gene that can be induced by both ischemia and stress. The results also suggest that inhibition of SMS1 mRNA expression may contribute to ceramide accumulation in a damaged cortex.

The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.

We have previously characterized the structure of the human MOB gene ( TMEM23 ), which encodes a hypothetical transmembrane protein (Vladychenskaya et al., 2002, 2004). The primary structure of the peptide that we predicted coincided completely with the amino acid sequence of the later identified sphingomyelin synthase 1 protein (SMS1), which catalyses the transfer of a phosphorylcholine moiety from phosphatidylcholine to ceramide, producing sphingomyelin and diacylglycerol (Huitema et al., 2004; Yamaoka et al., 2004). The gene we found was the SMS1 gene. The combination of in silico and RT-PCR data helped us identify and characterize numerous new transcripts of the human SMS1 gene. We identified mRNA isoforms that vary in the 5′-untranslated region (UTR) and encode the full-length protein, and transcripts resulting from alternative combinations of the exons in the coding region of the gene and the 3′-UTR. Comparison of the discovered transcripts' structures with the sequence of human chromosome 10 showed that the human SMS1 gene comprises at least 24 exons. RT-PCR and real-time PCR data showed that the expression patterns of the alternative SMS1 transcripts are tissue specific. Our results indicate that the regulation of SMS1 expression is complex and occurs at the transcriptional, post-transcriptional and translational levels.

Background The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex tissues to the action of Semax in vivo. Results The gene-expression alteration caused by the action of the peptide Semax was compared with the gene expression of the “ischemia” group animals at 3 and 24 h after permanent middle cerebral artery occlusion (pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and, 24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression. Among the immune-response genes, the expression of which was modulated by Semax, genes that encode immunoglobulins and chemokines formed the most notable groups. In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and vasculogenesis. Conclusions Semax affects several biological processes involved in the function of various systems. The immune response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote the formation and functioning of the vascular system. The immunomodulating effect of the peptide discovered in our research and its impact on the vascular system during ischemia are likely to be the key mechanisms underlying the neuroprotective effects of the peptide.