ITGB3 Research Articles

Preclinical In Vitro Model to Assess the Changes in Permeability and Cytotoxicity of Polarized Intestinal Epithelial Cells during Exposure Mimicking Oral or Intravenous Routes: An Example of Arsenite Exposure.

The gastrointestinal tract (GIT) is exposed to xenobiotics, including drugs, through both: local (oral) and systemic routes. Despite the advances in drug discovery and in vitro pre-clinical models, there is a lack of appropriate translational models to distinguish the impact of these routes of exposure. Changes in intestinal permeability has been observed in different gastrointestinal and systemic diseases. This study utilized one such xenobiotic, arsenic, to which more than 200 million people around the globe are exposed via their food, drinking water, work environment, soil, and air. The purpose of this study was to establish an in vitro model to mimic gastrointestinal tract exposure to xenobiotics via oral or intravenous routes. To achieve this, we compared the route (mimicking oral and intravenous exposure to GIT and the dose response (using threshold approach) of trivalent and pentavalent inorganic arsenic species on the permeability of in vitro cultured polarized T84 cells, an example of intestinal epithelial cells. Arsenic treatment to polarized T84 cells via the apical and basolateral compartment of the trans-well system reflected oral or intravenous routes of exposure in vivo, respectively. Sodium arsenite, sodium arsenate, dimethyl arsenic acid sodium salt (DMAV), and disodium methyl arsonate hydrate (MMAV) were assessed for their effects on intestinal permeability by measuring the change in trans-epithelial electrical resistance (TEER) of T-84 cells. Polarized T-84 cells exposed to 12.8 µM of sodium arsenite from the basolateral side showed a marked reduction in TEER. Cytotoxicity of sodium arsenite, as measured by release of lactate dehydrogenase (LDH), was increased when cells were exposed via the basolateral side. The mRNA expression of genes related to cell junctions in T-84 cells was analyzed after exposure with sodium arsenite for 72 h. Changes in TEER correlated with mRNA expression of focal-adhesion-, tight-junction- and gap-junction-related genes (upregulation of Jam2, Itgb3 and Notch4 genes and downregulation of Cldn2, Cldn3, Gjb1, and Gjb2). Overall, exposure to sodium arsenite from the basolateral side was found to have a differential effect on monolayer permeability and on cell-junction-related genes as compared to apical exposure. Most importantly, this study established a preclinical human-relevant in vitro translational model to assess the changes in permeability and cytotoxicity during exposure, mimicking oral or intravenous routes.

Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology

To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

Determination of the prevalence of thrombogenic risk factors on the examination of children in the Health Centre

Introduction. Thrombosis in children and adults is believed to be always multifactorial and caused by a combination of permanent and temporary thrombogenic risk factors. Carriage of hereditary or life-long thrombogenic risk factors accompanying a person, causing a tendency to occur arterial and venous thrombosis is a critical problem, but little studied in paediatrics. Materials and methods. A genetic study of twelve prothrombotic polymorphic variants of candidate genes was carried out at the Health Centre. Three hundred 96 children were examined; 177 (43.3%) were boys, and 219 (56.7%) were girls. The average age of the patients was 15.6 ± 1.8 years. The main group of children with thrombogenic risk factors consisted of 27 cases, including ten boys and 17 girls. The comparison group consisted of 396 adolescents, including 167 boys and 202 girls. Results. The study of genetic polymorphisms of the genes of blood clotting factors and genes of folate metabolism in the work of the Health Centre revealed the prevalence of the A2756G allele of the MTR gene (p = 0.032) and the C1565 allele of the ITGB3 gene (p = 0.012) in the population of girls to be statistically significantly higher than in boys. The proportion of the 4G allele (-675) of the PAI-1 gene (p = 0.028) was also determined to be significantly more often detected in boys, while the proportion of the 5G allele of the PAI-1 gene (p = 0.032) was found with a higher frequency in girls. The distribution of allele and genotype frequencies in the studied genes of blood coagulation factors and folate metabolism were checked for compliance with the Hardy-Weinberg equilibrium. The group of children with thrombogenic risk factors included 27 patients. In children of this group, in 64.3% of cases, a mutation of the GA genotype of the factor V Leiden gene was detected, and 37.5% of children were with compounds from the homozygous TT allele of the C677T MTHFR genotype and the homozygous AA allele of the G(-455)A genotype of the fibrinogen FBG gene. Conclusion. With the introduction of personalized prophylaxis in the thrombogenic risk group based on the children’s health centre, thrombogenic complications can be successfully prevented.

Distribution of Polymorphic Markers of Genes Encoding the Renin-angiotensin System (ACE, AGT, AGTR1), ITGB3, PPARG, Pparg in Patients With Essential Hypertension Depending on the Age

Essential hypertension (EH) is a multifactorial disease with a hereditary predisposition. Genes encoding the renin-angiotensin system (RAS) play a leading role in the stabilization of elevated BP in the course of EH, which determines the relevance of our clinical and genetic study. The study was based on the determination of the frequencies of polymorphic markers of the AGT, AGTR1, ACE, ITGB3, PPARG genes in 2 groups, depending on the age of patients (up to 60 years - group1, n=18, and after 60 years - group2, n=31), for the intergroup frequencies comparison and comparison of group frequencies with population data. Genotyping by gene polymorphisms was performed by real-time polymerase chain reaction. 24-hour ambulatory BP monitoring (ABPM) and Holter HR monitoring were conducted. Phenotypic features of the course of EH were accompanied by changes in the frequency characteristics of the studied genotypes. In patients of group 1, an increase in the frequency of protective genotypes of the ACE(II) and ITGB3(TT) genes was observed (p=0.004;0.015), which confirms the hypothesis of a possible favorable course of EH in patients under 60 years.

Integrative system biology and mathematical modeling of genetic networks identifies shared biomarkers for obesity and diabetes.

Obesity and type 2 and diabetes mellitus (T2D) are two dual epidemics whose shared genetic pathological mechanisms are still far from being fully understood. Therefore, this study is aimed at discovering key genes, molecular mechanisms, and new drug targets for obesity and T2D by analyzing the genome wide gene expression data with different computational biology approaches. In this study, the RNA-sequencing data of isolated primary human adipocytes from individuals who are lean, obese, and T2D was analyzed by an integrated framework consisting of gene expression, protein interaction network (PIN), tissue specificity, and druggability approaches. Our findings show a total of 1932 unique differentially expressed genes (DEGs) across the diabetes versus obese group comparison (p≤0.05). The PIN analysis of these 1932 DEGs identified 190 high centrality network (HCN) genes, which were annotated against 3367 GO terms and functional pathways, like response to insulin signaling, phosphorylation, lipid metabolism, glucose metabolism, etc. (p≤0.05). By applying additional PIN and topological parameters to 190 HCN genes, we further mapped 25 high confidence genes, functionally connected with diabetes and obesity traits. Interestingly, ERBB2, FN1, FYN, HSPA1A, HBA1, and ITGB1 genes were found to be tractable by small chemicals, antibodies, and/or enzyme molecules. In conclusion, our study highlights the potential of computational biology methods in correlating expression data to topological parameters, functional relationships, and druggability characteristics of the candidate genes involved in complex metabolic disorders with a common etiological basis.

ITGB1 promotes apoptosis in H9C2 cells

Myocardial infarction is a heart disease with high mortality rate. The research on the cardiomyocyte apoptosis is helpful to the prevention and treatment of myocardial infarction. As a cell surface receptor, the function of ITGB1 gene in cardiomyocytes is not clear. This study aimed to analyze the function and molecular mechanism of cardiomyocyte derived ITGB1 in myocardial apoptosis. To analyze cardiomyocyte apoptosis, TUNEL staining was performed with ITGB1 overexpression and knockdown in H9C2 cells. Apoptosis-related markers were also detected using Western blot. In addition, apoptosis-related signaling pathways were detected to elucidate the molecular mechanism regulated by ITGB1. Our results indicated that ITGB1 promoted cardiomyocyte apoptosis by inhibiting the Notch1 and Smad2/3 signal pathways. These data highlight the potential usefulness of ITGB1 as a potential target for preventing myocardial infarction.

IDENTIFICATION OF FERTILITY HAPLOTYPES IN THE BELARUSIAN POPULATION OF HOLSTEIN CATTLE

Long-term artificial selection of highly productive breeding animals, latent carriers of genetic diseases, has led to the accumulation of recessive mutations in the cattle population. Since 2007, the Laboratory of Animal Genetics has been researching the fertility haplotypes of Holstein cattle (HHC, HHB, and HHD), and since 2016, the Laboratory has started additional research by other haplotypes (HH0, HH1, HH3, HH4, HH5, HCD) that affect reproductive traits and are associated with embryonic and early postembryonic death of calves. Earlier developed methods allowed us to identify mutations in the FANCI, APAF1, SMC2, GART, TFB1M, SLC35A3, ITGB2, APOB, and UMPS genes associated with these haplotypes. Monitoring cattle populations in Belarus (n = 4101 heads) made it possible to reveal the frequency of occurrence of the hidden carriers of mutant alleles of the HH0 haplotype amounting to 3.42%, HH1 — 2.82%, HH3 — 3.75%, HH4 — 0.59%, HCD — 2.35%, HH5 — 2.2%, HHC — 2.56%, HHB — 0.65%, and HHD — 0%. We believe that further DNA diagnostics of foreign and domestic breeding will allow diminishing the spread of genetic defects that degrade the reproductive qualities of cattle.

Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. DisclosuresNo relevant conflicts of interest to declare.

A Phase 1/2 Study of Lentiviral-Mediated Ex-Vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I): Interim Results

▪Background: Leukocyte Adhesion Deficiency-I (LAD-I) is a rare disorder of neutrophil adhesion resulting from ITGB2 mutations encoding for the β2-integrin component, CD18. Severe LAD-I (CD18 <2% of polymorphonuclear [PMN] cells) is characterized by severe infections, impaired wound healing, and childhood mortality. Allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative; however, its efficacy is limited by donor availability and risk of graft-versus-host disease (GVHD) and graft failure. RP-L201-0318 (NCT03812263), a phase 1/2 open-label trial currently underway, employs autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the ITGB2 gene (cDNA)Methods: Pediatric patients ≥ 3 months old with severe LAD-I are eligible. HSCs are collected via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor and transduced with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning precedes RP-L201 infusion. Patients are followed for safety and efficacy (i.e., survival to age 2 and at least 1-year post-infusion, increase in PMN leukocyte CD18 expression to at least 10%, peripheral blood [PB] vector copy number [VCN], decrease in infections/hospitalizations, and resolution of skin or periodontal abnormalities).Results: Seven patients (ages 5mos-9yrs) have received RP-L201; all with follow-up ≥3 to 18 months. RP-L201 cell doses ranged from 2.8x106 to 6.5x106 CD34+ cells/kg with VCN from 1.8-3.8 copies/cell. No serious RP-L201 related treatment-emergent adverse events were reported. PB PMN CD18 expression in Patient 1 at 18-months post-treatment was 40% (vs. < 1% at baseline), with PB VCN of 1.44. Baseline skin lesions resolved with no new lesions reported. CD18 expression in the subsequent 6 patients has been 25-80% in neutrophils and stable for each patient from 3 to up to 18 months post-treatment. No new infections have been reported in patients post-infusion. The safety profile of RP-L102 remains highly favorable with no serious adverse events (SAEs) attributed to the investigational product (IP).Conclusion: RP-L201 leads to durable neutrophil CD18 expression and improved clinical course. Additional patient treatment is ongoing in 2021. DisclosuresKohn: UC Regents: Patents & Royalties; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees. Booth: GSK: Honoraria; Takeda: Honoraria; SOBI: Consultancy, Honoraria; Orchard Therapeutics: Consultancy, Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Almarza: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. de Oliveira: Orchard Therapeutics: Research Funding; Bluebird Bio: Research Funding. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

СВЯЗЬ ПОЛИМОРФИЗМА ГЕНОВ ТРОМБОЦИТАРНЫХ РЕЦЕПТОРОВ GPIA(С807Т), GPIIIA (Т1565С) С АКТИВНОСТЬЮ ТРОМБОЦИТОВ И ЭФФЕКТИВНОСТЬЮ АЦЕТИЛСАЛИЦИЛОВОЙ КИСЛОТЫ У ПАЦИЕНТОВ СО СТАБИЛЬНОЙ СТЕНОКАРДИЕЙ НАПРЯЖЕНИЯ

Background. Recently, much attention has been paid to the genetic aspects of thrombus formation. Understanding the genetic contribution to platelet function may have clinical implications for personalized pharmacotherapy. The aim of the study was to assess the distribution of polymorphic variants C807T of the ITGA2 gene and T1565C of the ITGB3 gene and to study their effect on platelet activity and acetylsalicylic acid (ASA) efficiency in patients with stable angina (SA) living in the Grodno region. Material and methods. The study included 92 patients with SA, 89 of them underwent elective percutaneous coronary intervention (PCI), and 93 were apparently healthy people. The survey data (general clinical, aggregometry, general blood count and platelet indices, polymerase chain reaction genotyping) were analyzed using the STATISTICA 10.0 program. Results. The prevalence of carriage of genotypes associated with possible variability of response to ASA therapy among patients with SA was 69.6% for the C807T polymorphic locus of the ITGA2 gene, 31.5% for the T1565C polymorphic ITGB3 locus, 60.2% and 37.6% among practically healthy individuals respectively. A lower frequency of the CC variant of the C807T polymorphic locus of the ITGA2 gene was noted in the group of men with SA in comparison with the control group of men (p = 0.043) according to Fisher's exact test. The patients were divided into subgroups (SG). Carriers of the CC genotype of the C807T polymorphic locus of the ITGA2 gene and TT genotype of the T1565C polymorphic locus of the ITGB3 gene were in SG1. Carriers of the CT+TT genotypes of the C807T polymorphic locus of the ITGA2 gene and TC+CC of the T1565C polymorphic locus of the ITGB3 gene were in SG2. The ASPI-test values were 23.0 [14.0; 50.5] U in SG1 and 22.5 [14.5; 34.5] U in SG2 of the ITGA2 gene, p&gt;0.05. The ASPI-test values were 23.0 [16.0; 38.0] U in SG1 and 20.0 [14.0; 34.0] U in SG2 of the ITGB3 gene, p&gt;0.05. Conclusions. No relationship was found between the polymorphic variants C807T of the ITGA2 gene and T1565C of the ITGB3 gene and platelet activity and the effectiveness of ASA in patients with SA.

Analysis of the association of polymorphisms of genes markers functions of endothelium and vascular-plate hemostasis with development of diabetic foot syndrome

The aim. To study the prevalence of various combinations of polymorphic variants of genes markers of endothelial function and vascular-platelet hemostasis in the development of diabetic foot syndrome.Materials and methods. In 198 patients with uncomplicated diabetes mellitus and 199 patients with diabetic foot syndrome, the frequency of polymorphic variants of the NOS 786C&gt;T, END1 Lys198Asn, ITGB3 1565T&gt;C (Leu33Pro), F5 1691G&gt;A, F2 20210G&gt;A, MMP9 8202A&gt;G, MTHFR 1298A&gt;C, VEGFA-634C&gt;G genes was studied. Using binary logistic regression analysis, the relationship of various combinations of polymorphisms of the studied genes with the development of diabetic foot was assessed.Results. In diabetic foot syndrome, the most significant contribution is made by the combination of polymorphic variants of the ITGB3 1565T&gt;C (Leu33Pro) and MTHFR 1298A&gt;C genes. With the development of this complication of diabetes mellitus, a combination of the 1565 TC polymorphism of gene ITGB3 and the 1298AA polymorphism of gene MTHFR is 2.1 times more common. The association of the 1565TT polymorphism of gene ITGB3 and the 1298AC polymorphism of gene MTHFR is 2 times more common in diabetes mellitus without complications.Conclusion. The combination of the 1565TС polymorphism of gene ITGB3 and the 1298АА polymorphism of gene MTHFR is associated with the risk of developing a diabetic foot and increases the risk of developing this complication by 2.4 times. The presence of a combination of the 1565TT polymorphisms of gene ITGB3 and the 1298AC polymorphism of gene MTHFR is more common in uncomplicated diabetes mellitus, which suggests its protective effect against the development of diabetic foot syndrome.

Associations of the ITGB3 gene rs5918T&amp;gt;C and the APOA1 gene rs1799837C&amp;gt;T markers with serum lipid metabolism in coronary artery disease patients

Abstract Aim The relationship between hypercholesterolemia, particularly elevated low density lipoprotein-cholesterol (LDL-C) levels and coronary artery disease is recognized by the evidence from previous epidemiologic studies. Importantly, genetic polymorphisms on different genes have been reported to be associated with plasma lipid levels. In this particular study, we aimed to investigate the relationship between the ITGB3 gene rs5918 T&amp;gt;C and APO-A1 gene rs1799837 C&amp;gt;T markers and serum lipid metabolism. Patients and methods A total of 100 subjects with CAD and 250 healthy subjects were involved in the current study. A basic biochemical analysis, including serum glucose, total serum cholesterol, HDL-C, LDL-C and triglycerides, was performed for each participant. Genotyping for the ITGB3 gene and APO-A1 gene polymorphisms was performed by polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis. Results With respect to the genotype and allele distributions of ITGB3 rs5918 T&amp;gt;C polymorphism, the frequency of the C allele was higher in the coronary artery disease (CAD) group compare to control group (p=0.001). Moreover, there was a statistically significant association detected between ITGB3 rs5918 CC genotype and serum total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) (p=0.0006, p=0.016 respectively) in CAD group. However there was no statistically significant association was identified between the APOA1 rs1799837 C&amp;gt;T polymorphism and biochemical parameters in control and CAD group. Conclusion The results demonstrated that rs5918 T&amp;gt;C variant within the ITGB3 gene might have a clinical importance as a genetic marker which increases the susceptibility to CAD. Therefore, the ITGB3 gene rs5918 C allele may be offered as a screening option for CAD in Turkish Cypriot population who come in for medical check-up. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Near East University

РАСПРОСТРАНЕННОСТЬ ПОЛИМОРФИЗМОВ ГЕНОВ ФАКТОРОВ РЕГУЛЯЦИИ СОСУДИСТОГО ТОНУСА, РЕЦЕПТОРОВ ТРОМБОЦИТОВ, РЕМОДЕЛИРОВАНИЯ СОСУДИСТОЙ СТЕНКИ И ПРОТРОМБОТИЧЕСКИХ ФАКТОРОВ ПРИ СИНДРОМЕ ДИАБЕТИЧЕСКОЙ СТОПЫ

Objective. To analyze the frequency of occurrence of eNOS 786C&gt;T, END1 Lys198Ash, ITGB3 1565T&gt;C (Leu33Pro), F5 1691G&gt;A, F2 20210G&gt;A, MMP9 8202A&gt;G, MTHFRA 1298 C,VEGFA 634C&gt;G gene polymorphisms in diabetic foot syndrome. Methods. The study included patients (n=198) with uncomplicated diabetes mellitus and patients (n=199) with the development of diabetic foot syndrome. Genotyping of the studied gene polymorphisms was performed by the polymerase chain reaction method. Results. In patients with uncomplicated diabetes mellitus and in patients with diabetic foot, no significant differences were found in the frequency of occurrence of polymorphisms 786C&gt;T of the eNOS3 gene, Lys198Ash of the END1 gene, 1691G&gt;A of the F5 gene, 20210G&gt;A of the F2 gene, 8202A&gt;G of the MMP9 gene, 634C&gt; G of the VEGFA gene and their allelic variants. Associative relationship between the polymorphisms of the NOS 786C&gt;T, END1 Lys198Ash, F5 1691G&gt;A, F2 20210G&gt;A, MMP9 8202A&gt;G, VEGFA 634C&gt;G genes and the development of diabetic foot syndrome was not found. With the development of a diabetic foot, a heterozygous variant of the 1565TС polymorphism of the ITGB3 gene was more common than in uncomplicated diabetes mellitus (37.7% and 28.3%, respectively (χ²=6.243, p=0.045)). The risk of developing a diabetic foot with the carriage of this polymorphism is 1.5 times higher than with other variants of gene polymorphism ((OR=1.534 (95% Cl 1.006 - 2.338), p&lt;0.05). Polymorphism 1298 AS of the MTHFR gene was more often detected in the group of patients with uncomplicated diabetes mellitus than in the group with the development of diabetic foot (45.4% and 29.1%), (χ²=11.55, p=0.004). Conclusion. The development of diabetic foot syndrome is associated with the carriage of the heterozygous 1565TС polymorphism of the ITGB3 gene, in which the likelihood of developing this complication increases 1.5 fold. In diabetes mellitus without complications, a more frequent carriage of the heterozygous polymorphism 1298 AC of the MTHFR gene was revealed, which may have a protective effect against the development of diabetic foot syndrome. What this paper adds The frequency of occurrence of genetic polymorphisms for regulating vascular tone, platelet receptors, vascular wall remodeling and prothrombotic factors and their allelic variants in diabetic foot syndrome was studied. It was found that the frequency of heterozygous 1565TС polymorphism of the ITGB3 gene in the development of diabetic foot is 1.3 fold higher than in the uncomplicated course of the disease. It was revealed that the fregnency of this variant of the ITGB3 gene polymorphism is associated with the risk of developing this complication of diabetes mellitus. In the presence of the heterozygous 1565TС polymorphism of the ITGB3 gene, the risk of developing diabetic foot syndrome increases by 1.5 fold than with the carriage of other variants of the T1565С polymorphism of the ITGB3 gene. It was found that in the uncomplicated course of the disease, the heterozygous genotype 1298 AC of the MTHFR gene occurs 1.6 fold more often than in the development of diabetic foot syndrome.

Exposure to Bacteriophages T4 and M13 Increases Integrin Gene Expression and Impairs Migration of Human PC-3 Prostate Cancer Cells.

The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 107 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins ITGAV, ITGA5, ITGB1, ITGB3, and ITGB5. Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of ITGAV, ITGA5, ITGB3, ITGB5 genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies.

ITGB6 inhibits the proliferation of porcine skeletal muscle satellite cells.

The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors. Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain- or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.

Clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia

The aimof the study was to assess the clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia (SS).Materials and methods.A total of 100 patients with SS were examined and followed-up for 15.3±8.3 months. The patients were divided into subgroups (SG): SG1 (n=51) – persons without events, SG2 (n=49) persons with recurrent ischemic events (hospitalization due to the development of pain syndrome, re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes), SGB (n=11) – persons with «major» recurrent ischemic events (re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes) , SGG (n=89) – persons without «major» events. The obtained survey data (general clinical, aggregometry, polymor phism of genes of platelet fibrinogen receptor ITGB3 (T1565C), platelet collagen receptor ITGA2 (C807T), ADP platelet receptor P2RY12, H1/H2 (T744C)) were analyzed using the STATISTICA 10.0 software.Results.In SG2, men predominated (χ2=9.2; p&lt;0.01), past MI was more common (χ2=4.8; p&lt;0.05), more stents were implanted (2.4±1.9 versus 1.7±1.1, p&lt;0.05), TRAP-test values were higher (p&lt;0.05) compared to SG1. In SGB, greater number of stents were implanted (3.1±2.2 versus 1.61±1.57, p&lt;0.05), the carriage of the TC genotype of the ITGB3 gene was more common, (p&lt;0.05), a combination of gene mutations ITGB3 and P2RY12 was more common, (p&lt;0.05) compared to SGG. A logistic regression equation was constructed, including the presence of diabetes mellitus, the number of platelets in the blood test, the ASPI-test values, the carriage of the 1565C allele of the ITGB3 gene, the number of stents implanted, which makes it possible to determine the likelihood of developing «major» recurrent ischemic events with a cut-off threshold LP₀=0.0965, with sensitivity – 81.82 %, specificity – 78.48 %, overall accuracy – 78.89 %.Conclusions.The factors associated with the development of recurrent ischemic events are: male sex, previous MI, a greater number of implanted stents, and high TRAP-test values. The factors associated with the development of recurrent «major» ischemic events are: a greater number of implanted stents, carriage of the TC genotype of the ITGB3 gene, carriage of a combination of mutations of the H1/H2 polymorphic locus of the P2RY12 gene and the T1565C polymorphic locus of the ITGB3 gene, diabetes mellitus, the number of platelets in blood test, ASPI-test values.

The rs5918 polymorphism in the &lt;i&gt;ITGB3&lt;/i&gt; gene increases the risk for preeclampsia in pregnant women with fetal growth retardation

Aim. To assess the relationship of rs5918 ITGB3, rs1126643 ITGA2 and rs5985 F13A1 polymorphic loci with the risk for preeclampsia (PE) in pregnant women with fetal growth retardation (FGR).&#x0D; Materials and methods. The study included 272 pregnant women, of which 76 had a combination of PE and FGR and 196 had FGR. In the studied groups, genetic testing was carried out for three polymorphic loci of candidate genes for hereditary thrombophilia (rs5918 ITGB3, rs1126643 ITGA2, and rs5985 F13A1).&#x0D; Results. The rs5918 genetic variant in the ITGB3 gene is associated with the development of PE in pregnant women with FGR: C allele of rs5918 ITGB3 increases the risk for this complication of pregnancy by 1,8 times (OR 1.761.77, p0.036, pperm0.038). The rs5918 polymorphism determines an increase in the affinity of DNA motifs for seven transcription factors (BDP1, ELF1, IRF, NRSF, Pax-5, Sp1, and Zfx), is a missense mutation and causes the Leu59Pro amino acid substitution in the 3 subunit of integrin, is multidirectionally associated with the expression of five genes (EFCAB13, TBKBP1, NPEPPS, MRPL45P2, THCAT158) and alternative splicing of two genes (EFCAB13, MRPL45P2), is located in the region of functionally important DNA regions (promoters and enhancers) in cell cultures and organs which are pathogenetically important for the formation of PE and FGR.&#x0D; Conclusion. The rs5918 polymorphism in the ITGB3 gene increases the risk for PE in pregnant women with FGR.

Polymorphisms of F2 (G20210A), F5 (G1691A), F 7 (G10976A), F 13(G13T), FGB, ITGA2, ITGB3, PAI-I genes and its association with thrombotic complications in patients suffering from Takayasu aortoarteritis of Urals population.

Cardiovascular complications, especially thrombotic events, are characteristic for Takayasu arteritis (TA). These events significantly deteriorate the patients' quality of life and cause disability and preterm death. Coagulation factor II (F2, G20210A), coagulation factor V (F5, G1691A, Leiden), coagulation factor VII (F7, G10976A), coagulation factor XIII (F13, G13T), fibrinogen (FGB), platelet alpha subunit of transmembrane receptor for collagens and related proteins (ITGA2), platelet glycoprotein (ITGB3), and plasminogen activator inhibitor-1 (PAI-I) gene polymorphisms coexist with TA, and their pathophysiologic interaction needs to be studied. A total of 43 patients with TA were examined for nucleotides polymorphism in F2 (G20210A), F5 (G1691A, Leiden), F7 (G10976A), F13 (G13T), FGB, ITGA2, ITGB3, and PAI-I genes using polymerase chain reaction. Moreover, 130 sex- and age-adjusted healthy controls without a history of any thrombotic complications were enrolled. Among the patients with TA, there were 34 women aged between 17 and 77 (mean 49, median 49; Q1-Q3, 36-61) years and 9 men aged between 20 and 66 (mean 37.8, median 38; Q1-Q3: 31-45) years. Thrombotic complications were recorded in 22 (51%) patients with TA. Comparison of thrombophilia markers genotypes in patients with TA and healthy controls revealed homozygous and heterozygous mutation in ITGA2 (p<0.0001) and PAI-I genes (p=0.026). The frequency of occurrence of hereditary thrombophilia markers in patients with TA was assessed. Detection of the PAI-I gene mutation was significantly more frequent (p=0.032) in patients with TA with a history of thrombotic events than in those with no thrombosis history. Detection of multiple (more than 4 genes) simultaneous mutations of thrombophilia markers was significantly (p=0.0001) more frequent in patients with TA with a history of thrombotic events. Assessment of hereditary thrombophilia genetic markers reveals additional (genetic) risk markers of thrombotic complications in patients with TA and may help in decision making for antiplatelet and/or anticoagulant treatment in patients with TA to reduce the risk of thrombotic complications.

Association of ITGB3 gene polymorphisms with the risk of developing fetal growth restriction syndrome

The article presents a molecular genetic study of the ITGB3 gene polymorphism in pregnant women with fetal growth restriction syndrome. Purpose: Assessment of the detectability of the association of the ITGB3 gene polymorphism in women with physiological pregnancy and fetal growth restriction syndrome in the uzbek population of the Bukhara region. Materials and methods: 80 pregnant women were examined during gestation from 8-36 weeks: 40 pregnant women with fetal growth restriction syndrome and 40 women with physiological pregnancy, hospitalized in the department of pathology of pregnant women in the Bukhara city maternity hospital in the city of Bukhara. Conclusion: The study showed that the functionally unfavorable allele C and the association of the T / C genotype polymorphism of the ITGB3 gene polymorphism can be a determinant of an increased risk of developing FGRS in Uzbekistan (χ2 &lt;0.47; P&gt; 0.05).

Screening Candidate Genes Regulating Placental Development from Trophoblast Transcriptome at Early Pregnancy in Dazu Black Goats (Capra hircus).

Simple SummaryThe trophoblast is an original placental tissue whose normal proliferation, differentiation, migration, adhesion, and angiopoiesis are essential for placenta formation and fetal survival during early pregnancy. However, the key genes and molecular mechanisms involved in placenta development in goats are unknown. Herein, the morphology and histological structures of trophoblast tissues from day 20 to 30 of pregnancy were determined. RNA-sequencing was used to screen potential functional genes in common highly expressed and differentially expressed genes. RAP1 signaling pathway was used as the contact center and coordinated with other pathways to regulate placenta development. This study could provide insights into the molecular mechanisms underlying ruminant placentation.This study explored the trophoblast transcriptome to understand potential functional genes involved in early placental development in goats and their enriched signaling pathways. Trophoblast samples were collected from nine Dazu Black goats on days 20, 25, and 30 of pregnancy (D20, D25, and D30). As the pregnancy progressed, the morphology and histological structures showed significant growth, adhesion, and angiogenesis. A total of 23,253 commonly expressed genes (CEGs) and 4439 differently expressed genes (DEGs) were detected by RNA sequencing. The common highly expressed genes (ChEGs) (the top 100 CEGs) with the highest FPKM percentage (29.9%) of all CEGs were annotated to the ribosome pathway and maintain pregnancy. DEGs were abundant in D30 vs. D20 (3715 DEGs). Besides, the DEGs were associated with the inhibition of oxidative phosphorylation and activation of PI3K-Akt, focal adhesion, ECM–receptor interaction, Rap1, and CAM signaling pathways. The RAP1 may be a central pathway since it coordinates with others to regulate the cell proliferation, invasion, migration, and fusion of trophoblasts. qRT-PCR and Western blot analysis confirmed the transcriptional expression in IGF1, VEGFC, RAPGEF3, PIK3CA, AKT3, ITGB3, ITGA11, SPP1, NOS1, and ATP6V0B genes and protein levels in VEGF, RAPGEF3, and Akt. This is the first study of transcriptome profiling in goat placenta and provides diverse genetic resources for further research on placenta development.