Main Research Findings

1) Short peptides, such as vesugen, have induced expression of differentiation factors in aging cultures, indicating that the peptides elicit geroprotective effects.

 

Selected Data

1) Vesugen was defined as a KED peptide by the research team of Khavinson et. al; KED refers to the amino acid sequence, Lys-Glu-Asp. Recent research has examined the role of KED peptides, such as vesugen, in the treatment of Alzheimer’s disease. Vesugen has been found to have neuroprotective effects on the neuroplasticity and dendritic spine morphology in mouse-based models of Alzheimer’s disease [1]. Short peptides have been found to have a number of biological applications, and in vitro and in vivo studies report that their ability to treat Alzheimer’s is promising. While vesugen was originally isolated as a component of blood vessels, research quickly found that the peptide had several neuroprotective qualities, specifically in elderly individuals. KED peptides were also revealed to enhance mental state, neurological functioning of the central nervous system, and memory in patients experiencing depression, attention deficits, or memory impairments [1].

It is important to mention the correlation between mushroom spines and memory. Mushroom spines are postsynaptic structures that form most active synapses and contain a large amount of postsynaptic density receptors. Mushroom spines are also referred to as “spines of learning” or “memory spines” [5]. With Alzheimer’s disease the number of mushroom spines present in the hippocampal neurons tend to decrease. That being said, in a mouse-based model of Alzheimer’s disease, KED peptide was found to significantly increase the amount of mushroom spines in hippocampal neurons. Because of the role they play in neuroplasticity and long term potentiation, a reduction in mushroom spines indicates impairments in memory and learning [1].

2) The research team of Khavinson et. al examined the effects of short peptides, pancragen, bronchogen, and vesugen on the expression of various transcription factors. The transcription factors WEDC1, Hoxa3, and CXCL12 were found in cellular cultures obtained from samples of human lung, pancreatic cells, and fibroblasts of the prostate gland at different points during the aging process. WEDC1, Hoxa3, and CXCL12 all have the potential to act as differentiation factors while synthesis of factors WEDC1 and CXCL12 has been shown to decrease with age, resulting in hyperplastic processes.

The experiments were performed on embryonic cultures of acinar cells found in the pancreas and the bronchial epithelium, specifically of passages 1, 7, and 14. In this study the different passages represented the maturity level of the cultures, with passage 1 defined as young, passage 7 defined as mature, and passage 14 defined as aged. Similarly, cultures of human prostate fibroblasts were obtained and separated into passages 1, 4, and 7. These passages correspond to the same maturity levels as passages 1, 7, and 14 in the pancreatic acinar cells and bronchial epithelial cells. The cultures were treated with saline if included in the control group, or with peptide if they were in the experimental group. Bronchogen was administered to the bronchial epithelial cells, pancragen was administered to the pancreatic acinar cells, and vesugen was added to the prostatic fibroblast cultures. It is important to mention how the peptides were administered as initial studies found that short peptides not specific to the tissue had no effect on the expression of the transcription factors [4].

In order to prepare the pancreatic acinar cells, the cells were cultured in 5 ml DMEM and supplemented with L-glutamine, 1% penicillin-streptomycin, and 15% fetal calf serum. Bronchial epithelial cells were cultured in MEM and supplemented in the same solution of L-glutamine, 1% penicillin-streptomycin, and 15% fetal calf serum. Finailly, the prostatic fibroblasts were cultured in 24-23ll plates with 5% CO2 and supplemented with 10% ECS, 100 ug/ml gentamicin, 300 ug/ml L-glutamine, and 0.02 M HEPES buffer. Immunocytochemical analysis was used to examine the synthesis of transcription factors in the cell and how they respond to administration of the peptides. Immunocytochemical analysis was performed morphometrically using a computed-assisted microscopic image analysis program. Additionally, immunofluorescence confocal microscopy was also performed on cell suspensions. In order to observe changes in the expression of different signal molecules, cell smears were treated with primary antibodies related to the CXCL12 and WEDC1 transcription factors [4].

Discussion

1) The effect of the KED peptide on neurogenesis markers nestin and growth associated protein 43 (GAP43) was observed using confocal microscopy and Western blot analysis. This is important to mention considering that nestin is typically expressed at the beginning stages of neuronal differentiation. That being said, expression of nestin in hippocampal cells was shown to decrease in mice with Alzheimer’s Disease. Furthermore, GAP43 tends to be expressed during the process of neuronal differentiation and is involved in the transmission of nerve impulses. There is a distinct correlation between the expression of GAP43 and initial stages of Alzheimer’s disease. The research team found that the peptide caused a 1.8-2.0-fold increase in the expression of nestin and GAP43.

The KED peptide was also assessed to determine its effects on transcription factors p16 and p21. Both of these factors have been shown to inhibit the activity of cyclin-dependent kinases. This results in the prevention of phosphorylation of the retinoblastoma protein responsible for regulating the cell cycle. Expression of transcription factors p16 and p21 in various organs, neurons, and liver cells was linked to age-related decline and reduced life expectancy. However, the research team found that expression of p16 is typically activated in cases of normal aging, while the expression of p21 is related to age-associated diseases such as Alzheimer’s. This was represented in the study as the mice with Alzheimer’s experienced increases in the expression of these protein markers. That being said, results of real-time PCR and immunofluorescence confocal microscopy testing found that administration of the KED peptide resulted in a 1.8-3.2-fold decrease in the expression of transcription factors p16 and p21 [1].

Finally, the response in expression of the genes SUMO1, APOE, and IGF1 to administration of the KED peptide was assessed in human fetal bone marrow mesenchymal stem cells. Both the SUMO1 and APOE genes experienced a decline in expression during the aging process. Small ubiquitin-like modifier 1 (SUMO1) is a protein involved in post-translational modification as it relates to the proteins that regulate transportation, transcription, apoptosis, and the phases of the cell cycle. Reduced expression and synthesis of the SUMO1 protein leads to decreased synaptic plasticity and memory deficits, thus indicating the crucial role it plays in the management of Alzheimer’s disease [1].

APOE is a blood plasma protein involved in the transportation of the cholesterol and triglyceride in metabolites. Additionally, this protein is involved in the restoration and proliferation of neurons. Dysfunction of this protein can lead to the accumulation of neurotoxic peptide A-beta-42 in the brain tissue. Insulin-like growth factor-1 (IGF-1) is a compound that assists in the proliferation and differentiation of many different types of cells due to its involvement throughout the endocrine, autocrine, and paracrine systems. Decreased levels of IGF-1 are linked to the development of metabolic syndrome and Alzheimer’s disease. Similar to APOE proteins, IGF-1 plays an important part in activating signaling cascades that help to prevent the accumulation of the neurotoxic peptide A-beta-42. The research team also discovered that exogenous administration of IGF-1 helped to normalize synaptic plasticity in cases of Alzheimer’s disease [1].

The KED peptide was able to stimulate expression of the SUMO1 and APOE genes by 1.2 and 2.2 times, respectively. The peptide was also shown to increase the expression of the IGF1 gene. Prior to administration of the KED peptide, IGF1 gene expression was reduced by 3- and 2-fold during replicative and stationary aging, respectively. Analysis of the data gathered by the research team indicated that the KED peptide works by binding to nucleosome fragments in order to regulate the expression of genes primarily responsible for apoptosis and aging. Normalization of the expression of the aforementioned genes can prevent the development of diseases like Alzheimer’s by preventing a pathogenic cascade of events [1].

2) Results of the study conducted by the research team of Khavinson et. al found that in all control groups of pancreatic acinar cells and bronchial epithelial cells there was verified expression of CXCL12 and Hoxa3 transcription factors. In the control group of prostatic fibroblast cell cultures there was verified expression of CXCL12 and WEDC1 transcription factors. In the acinar cell cultures, Hoxa3 expression was found to significantly reduce as the samples age. Expression of the transcription factor was marked as 1.2- and 2-fold greater in young cultures than mature and aged cultures, respectively. This is compared to CXCL12 expression that was shown to decrease by 22 and 12% in mature and aged cultures, respectively. Finally, in comparison to young cultures, mature and aged prostatic fibroblast cultures experienced a 60 and 81% respective reduction in WEDC1 expression [4].

After pancragen was added to the cultures of pancreatic acinar cells, the expression of CXCL12 decreased by 21% in young cultures, 60% in mature cultures, and 81% in aged cultures. Administration of pancragen resulted in a significant increase in expression of Hoxa3, with a 60 and 90% increase in young and mature cultures respectively. However, the most significant effect was elicited in aged cultures that experienced a 2.8-fold increase in Hoxa3 expression in comparison to the control. Administration of bronchogen to the bronchial epithelial cell cultures resulted in a similar increase in the expression of Hoxa3 transcription factors. In young, mature, and aged cultures expression increased by 1.7, 1.4, and 1.7 times, respectively. It is important to mention that administration of bronchogen to the bronchial epithelial cultures elicit no significant changes in the expression of CXCL12. Addition of vesugen to the prostatic fibroblast cultures resulted in increased expression of both CXCL12 and WEDC1. In young, mature, and aged cultures CXCL12 expression increased by 1.2, 8, and 7.6 times the control group, while WEDC1 expression increased by 1.5, 8.2, and 16 times the control group, respectively [4].

The increased expression of transcription factors indicates that short peptides such as pancragen, bronchogen, and vesugen have geroprotective properties. This is because aging of cells, specifically in the bronchial epithelium, pancreatic acinar cells, and prostate fibroblasts, are associated with significant reductions in the levels of CXCL12, Hoxa3, and WEDC1. Decreased expression typically results in a reduced differentiation capacity and functional activity of the bronchioles, pancreas, and prostate throughout the aging process.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Khavinson VK, Lin’kova NS, Umnov RS. Peptide KED: Molecular-Genetic Aspects of Neurogenesis Regulation in Alzheimer’s Disease. Bull Exp Biol Med. 2021 May;171(2):190-193. doi: 10.1007/s10517-021-05192-6. Epub 2021 Jun 26. PMID: 34173097.

[2] Kozlov KL, Bolotov II, Linkova NS, Drobintseva AO, Khavinson VK, Dyakonov MM, Kozina LS. [Molecular aspects of vasoprotective peptide KED activity during atherosclerosis and restenosis]. Adv Gerontol. 2016;29(4):646-650. Russian. PMID: 28539025.

[3] Apostolopoulos V, Bojarska J, Chai TT, Elnagdy S, Kaczmarek K, Matsoukas J, New R, Parang K, Lopez OP, Parhiz H, Perera CO, Pickholz M, Remko M, Saviano M, Skwarczynski M, Tang Y, Wolf WM, Yoshiya T, Zabrocki J, Zielenkiewicz P, AlKhazindar M, Barriga V, Kelaidonis K, Sarasia EM, Toth I. A Global Review on Short Peptides: Frontiers and Perspectives. Molecules. 2021 Jan 15;26(2):430. doi: 10.3390/molecules26020430. PMID: 33467522; PMCID: PMC7830668.

[4] Khavinson VKh, Linkova NS, Polyakova VO, Kheifets OV, Tarnovskaya SI, Kvetnoy IM. Peptides tissue-specifically stimulate cell differentiation during their aging. Bull Exp Biol Med. 2012 May;153(1):148-51. doi: 10.1007/s10517-012-1664-1. PMID: 22808515.

[5] Khavinson V, Ilina A, Kraskovskaya N, Linkova N, Kolchina N, Mironova E, Erofeev A, Petukhov M. Neuroprotective Effects of Tripeptides-Epigenetic Regulators in Mouse Model of Alzheimer’s Disease. Pharmaceuticals (Basel). 2021 May 27;14(6):515. doi: 10.3390/ph14060515. PMID: 34071923; PMCID: PMC8227791.

 

PEPTIDES PREFER THE COLD
Keep peptide vials refrigerated at all times to reduce peptide bond breakdown. DO NOT FREEZE. Most peptides, especially shorter ones, can be preserved for weeks if careful.
Always swab the top of the vial with an alcohol wipe, rubbing alcohol or 95% ethanol before use.
Before drawing solution from any dissolved peptide vial, fill the pin with air to the same measurement you will be filling with solution, ie. if you plan to take 0.1 ml, first fill the pin with 0.1ml of air, push the air into the vial, and then draw the peptide back up to the 0.1 ml marker. Doing so will maintain even pressure in the vial. Always remember to remove air bubbles from the pin by flicking it gently, pin side up, and pushing bubbles out. In addition, push out a tiny amount of solution to ensure there is no air left in the metal tip.

ONLY MIX WITH STERILE BACTERIOSTATIC WATER
The purity and sterility of bacteriostatic water are essential to prevent contamination and to preserve the shelf-life of dissolved peptides.
Push the pin through the rubber stopper at a slight angle, so that you inject the bacteriostatic water toward the inside wall of the vial, not directly onto the powder.
Lyophilized peptide should be stored at -20°C (freezer), and the reconstituted peptide solution at 4°C (refrigerated). Do not freeze once reconstituted.
NEVER SHAKE A VIAL TO MIX.

Air bubbles are unfavorable to the stability of proteins.

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