Recombinant Human Angiogenin Research Articles

МИКРОБТЫҚ, ҚАБЫНУҒА ҚАРСЫ ЖӘНЕ ЖАРАЛАРДЫ ЕМДЕУ ӘСЕРІН ЖАҚСАРТУ ӘДІСІ РЕТІНДЕГІ АДАМНЫҢ РЕКОМБИНАНТТЫ АНГИОГЕНИНІНІҢ НЕГІЗІНДЕГІ ЖАҚСАРТЫЛҒАН ГЕЛЬ

Modification of a recombinant gel based on angiogenin by adding dioxidin to its composition leads to a pronounced antimicrobial effect against the main pathogens of purulent-septic infections - Staphylococcus aureus, colostrum, late blight and E. coli. The purpose of this study: to study the antimicrobial, anti-inflammatory and wound healing effects of angiogenin gel in comparison with a modified version of this drug - dioxidin-angiogenin gel. Methods. The objects of research are natural recombinant human angiogenin gel and modified recombinant human angiogenin gel with dioxydine. Native angiogenin gel is intended for external use, contains recombinant human angiogenin at a concentration of 25 micrograms / ml, excipients-polyethylene oxide (filler) and sodium benzoate (preservative). Recombinant human angiogenin gel with modified dioxidin contains recombinant human angiogenin at a concentration of 25 micrograms/ml and 5% dioxidin, excipients – polyethylene oxide (filler) and sodium benzoate (preservative). The antimicrobial activity of the objects of study was studied by diffusion into agar. Reference strains from the Republican Collection of Microorganisms were used as test strains: gram-positive cocci-Staphylococcus aureus ATCC 25922; Pseudomonas aeruginosa ATCC 27853; gram-positive bacteria - Bacillus subtilis 8232 and gram-negative rods. The results of the study of the anti-inflammatory activity of angiogenin gel and modified dioxydin angiogenin gel, which gave positive results with local treatment. Conclusions. When modifying a gel based on recombinant angiogenin with the addition of a chemotherapeutic drug, dioxidin is used to treat staphylococcus, peristalsis, hay and E.coli, a pronounced antimicrobial effect is manifested.

Wound healing and anti-inflammatory effects of recombinant human angiogenin

New effective wound healing agents are a priority for modern clinical pharmacology. A promising approach would be to develop medicinal products that promote angiogenesis, which is a critical step in wound healing. The aim of the study was to evaluate the wound healing effect of a medicinal product based on recombinant human angiogenin in gel form in various experimental models. Materials and methods: white outbred male rats were used as experimental ani mals. The study compared healing effects of a regenerating product containing recombinant human angiogenin (0.0025%) in gel form and a reference product in full-thickness excision, incision, and burn wound models. The healing effect of the test product in treating chronic wounds was assessed in a model of alloxan-induced diabetes mellitus. The anti-inflammatory effect of the test product containing recombinant human angiogenin was compared with that of another reference product in a model of adjuvant-induced arthritis. Results: according to the study, the test product based on recombinant human angiogenin exerts higher wound healing effect in treating excision, incision, and burn wounds than the reference product (Solcoseryl gel). Being applied, the test product intensifies tissue repair in chronic wounds in the model of alloxan-induced diabetes. The dissociation of necrotic tissues and the progression towards epithelialisation at wound edges are more rapid. The anti-inflammatory effect of the test product based on recombinant human angiogenin is comparable with that of the reference product (Diclofenac gel). Conclusions: the test product based on recombinant human angiogenin in gel form was found to have pronounced wound healing and anti-inflammatory effects comparable with those of reference products.

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Исследование общей и специфической токсичности рекомбинантного ангиогенина человека

Angiogenin exerted no toxic effects in acute toxicity tests, all animals successfully endured the maximally attainable dose 12 g/kg intragastrically. To assess the long-term/chronic toxicity, the gel-based composition containing 0.01 % of angiogenin was daily applied to the scarified skin areas of rats for 1 month. No significant changes or differences between the treated and control groups were detected by common biochemical, hematological and histological assays. No local irritating effects of 0.0025 % angiogenin gel were detected in appropriate tests using outbred chinchilla rabbits. Angiogenin did not cause neither sensibilization of guinea pigs in active cutaneous anaphylaxis test nor delayed hypersensitivity reaction in mice. No detectable immuno- and embryotoxicity of the preparation were observed. Angiogenin was proved to lack mutagenic and carcinogenic capabilities, as well as the capability of stimulating the tumor growth upon intratumoral administration. The tested preparation of angiogenin showed no toxic effects, including systemic, local irritating, sensibilizing and skin-resorptive ones, and, thus, recombinant human angiogenin is a safe pharmacological substance. 10.30906/2073-8099-2022-14-2-40-53

Plant-derived angiogenin fusion protein's cytoprotective effect on trabecular meshwork damage induced by Benzalkonium chloride in mice.

BackgroundBenzalkonium chloride (BAK), commonly used in glaucoma treatment, is an eye drop preservative with dose-dependent toxicity. Previous studies have observed the multi-functional benefits of angiogenin (ANG) against glaucoma. In our study, we evaluated ANG’s cytoprotective effect on the trabecular meshwork (TM) damage induced by BAK. Additionally, we developed a plant-derived ANG fusion protein and evaluated its effect on TM structure and function.MethodsWe synthesized plant-derived ANG (ANG-FcK) by fuzing immunoglobulin G’s Fc region and KDEL to conventional recombinant human ANG (Rh-ANG) purified from transgenic tobacco plants. We established a mouse model using BAK to look for degenerative changes in the TM, and to evaluate the protective effects of ANG-FcK and Rh-ANG. Intraocular pressure (IOP) was measured for 4 weeks and ultrastructural changes, deposition of fluorescent microbeads, type I and IV collagen, fibronectin, laminin and α-SMA expression were analyzed after the mice were euthanized.ResultsTM structural and functional degeneration were induced by 0.1% BAK instillation in mice. ANG co-treatment preserved TM outflow function, which we measured using IOP and a microbead tracer. ANG prevented phenotypic and ultrastructure changes, and that protective effect might be related to the anti-fibrosis mechanism. We observed a similar cytoprotective effect in the BAK-induced degenerative TM mouse model, suggesting that plant-derived ANG-FcK could be a promising glaucoma treatment.

Effect of a gel based on recombinant human angiogenin on the healing of donor palate wounds

The aim of the study was to study the effect of the gel on the basis of recombinant human angiogenin on the rate of regeneration of donor palatal wounds. The study involved 20 patients (8 men and 12 women) aged 32 to 55 years. Patients were divided into two groups: the 1st group is a study group (n=10), whose patients in the postoperative period used a gel based on recombinant human angiogenin, the 2nd group is a control group (n=10) in which a gel based on recombinant human angiogenin was not used. Patients in both study groups underwent vestibuloplasty with simultaneous plasty of the attached keratinized gingiva with a free gingival graft from the area of the hard palate. The operations were carried out at the stage of disclosing dental implants, simultaneously with the installation of healing abatements or 4 weeks before dental implantation. For histological examination, tissue samples were obtained from the region of the edge of the donor's wounds of the palate at the 7th and 14th days after surgery. As a result of the study, significant differences were found in the comparison groups when assessing the processes of inflammation, angiogenesis and epithelization. The local application of the gel containing recombinant human angiogenin resulted in a rapid decrease in the intensity of inflammation in lamina propria mucosae and a significant decrease in the bulk density of cell infiltrates, accelerating regeneration. This is primarily due to the stimulation of the development of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and increased blood supply to the affected area, as well as an increase in the proportion of fibroblasts. The most important observation was the increase in the rate of epithelialization of donor wounds of the hard palate.

Modified gel based on recombinant human angiogenin: study of antimicrobial, anti-inflammatory and wound healing effects

Modified gel based on recombinant human angiogenin: study of antimicrobial, anti-inflammatory and wound healing effects

Apatinib Inhibits Angiogenesis Via Suppressing Akt/GSK3β/ANG Signaling Pathway in Anaplastic Thyroid Cancer

Background/Aims: Anaplastic thyroid carcinoma (ATC) is one of the most lethal human malignancies, and there is no efficient method to slow its process. Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma patients. However, the effects of Apatinib in ATC are still unknown. Methods: In this study, we explored the effects and mechanisms of Apatinib on tumor growth and angiogenesis in vitro and in vitro in ATC cells. Angiogenesis antibodies array was utilized to detect the expression of angiogenesis-related genes after Apatinib treatment in ATC cells. In addition, we used Akt activator, Akt inhibitor and GSK3β inhibitor to further study the mechanism for how Apatinib suppressed angiogenesis. Results: Apatinib treatment could suppress the growth of ATC cells in a dose- and time-dependent manner via inducing apoptosis and blocking cell cycle progression at G0/G1 phase. Moreover, Apatinib treatment decreased the expression of angiogenin (ANG) and inhibited angiogenesis of ATC cells in vitro and in vitro. We further confirmed that recombinant human ANG (rhANG) significantly abrogated Apatinib-mediated anti-angiogenic ability in ATC cells. Additionally, Apatinib treatment decreased the level of p-Akt and p-GSK3β. Moreover, the Apatinib-mediated decrease of ANG and anti-angiogenic ability were partly reversed when an Akt activator, SC79, was administered. Furthermore, the anti-angiogenic ability of Apatinib can be enhanced in the presence of Akt inhibitor, and the inhibition of GSK3β attenuated the anti-angiogenic ability of Apatinib. Conclusion: Our results demonstrated that Apatinib treatment inhibited tumor growth, and Apatinib-induced suppression of Akt/GSK3β/ANG signaling pathway may play an important role in the inhibition of angiogenesis in ATC, supporting a potential therapeutic approach for using Apatinib in the treatment of ATC.

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.

Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs

BackgroundMicrovascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses.MethodsC3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs.ResultsIn the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy.ConclusionsOur data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.

Evidence for the presence of angiogenin in human testis.

We have reported the expression and possible roles of angiogenin, a potent angiogenic factor, in human female reproductive organs. In this study, we investigated the expression of angiogenin in the human testis, a male reproductive organ. Western blot analysis showed the presence of angiogenin in the human testis, with a single band of the same size as recombinant human angiogenin. Immunohistochemical study and in situ hybridization showed that the angiogenin protein and messenger RNA (mRNA) localized in peritubular myoid cells (PTMCs) and vascular endothelial and smooth muscle cells. PTMCs are known to play various roles in the testes concerned with spermatogenesis, transport of spermatozoa, structural support to the seminiferous tubules, and mediation of Sertoli cell function. The specific localization of angiogenin in PTMCs suggests that angiogenin plays physiologic roles in the human testis.

Chemo-enzymatic synthesis of the human angiogenin gene. Construction of bacterial strains, producers of human angiogenin. Elaboration of a technology for the purification and preparation of angiogenin

Chemo-enzymatic synthesis of the gene of human angiogenin, a stimulator of blood vessel growth, was performed for the first time. Cloning of the human angiogenin gene in several expressing vector systems was carried out, and several bacterial strains, producers of human angiogenin were constructed. In particular, strains, producers of “angiogenin-β-galactosidase” and “angiogenin-domains of staphylococcal protein A hybrid proteins” were obtained, as well as a bacterial strain efficiently expressing free angiogenin. Large-scale preparation of purified recombinant human angiogenin was accomplished using theE.coli JM103 pRITA16 producer strain constructed, and the biological activity of the resulting preparation was studied.

Crystallization and preliminary X-ray analysis of human angiogenin

Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222 1 ( a = 83.36 A ̊ , b = 120.64 A ̊ , c = 37.72 A ̊ ) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution and are suitable for three-dimensional X-ray structural analysis.