Endothelial Gene Research Articles

Lysophospholipid (LPA) receptors in GtoPdb v.2023.1

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [62, 23, 91, 144]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [46], This discovery represented the beginning of the de-orphanisation of members of the endothelial differentiation gene (edg) family, as other LPA and sphingosine 1-phosphate (S1P) receptors were found. Five additional LPA receptors (LPA2,3,4,5,6) have since been identified [91] and their gene nomenclature codified for human LPAR1, LPAR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and Lpar1, Lpar2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function of their corresponding proteins. The crystal structure of LPA1 [17, 80, 2] and LPA6 [128] are solved and indicate that LPA accesses the extracellular binding pocket, consistent with its proposed delivery via autotaxin [17]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The binding affinities to LPA1 of unlabeled, natural LPA and anandamide phosphate (AEAp) were measured using backscattering interferometry (pKd = 9) [92, 115]. Utilization of this method indicated affinities that were 77-fold lower than when measured using radioactivity-based protocols [143]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Multiple groups have independently published validation of all six LPA receptors described in these tables, and further validation was achieved using a distinct read-out via a novel TGFα "shedding* assay [54]. LPA has been proposed to be a ligand for GPR35 [103], supported by a study revealing that LPA modulates macrophage function through GPR35 [60]. However chemokine (C-X-C motif) ligand 17 (CXCL17) is reported to be a ligand for GPR35/CXCR8 [85]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channels TRPV1 [96] and TRPA1 [65]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors.

Epigenetic landscape reveals MECOM as an endothelial lineage regulator

A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Abstract 4584: Genetic disruption of vascular endothelial profilin-1 impacts tumor microenvironment supressing tumorigenicity of renal cancer

Abstract Renal cell carcinoma (RCC) is estimated to result in 79,000 new cases and 13,920 deaths in 2022. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC and is characterized by a highly vascularized tumor microenvironment (TME). While current anti-angiogenic therapies targeting VEGF signaling are initially effective, almost all patients develop resistance to these therapies. Therefore, there is a need to identify clinically relevant alternative molecular targets to suppress tumor angiogenesis and progression in ccRCC. We previously discovered transcriptional upregulation of actin-binding profilin1 (Pfn1) in tumor-associated vascular endothelial cells (EC) in ccRCC and higher expression of Pfn1 correlated with adverse clinical prognosis. The goal of the present work was to further explore whether genetic manipulation of Pfn1 specifically in EC has therapeutic benefit in kidney cancer. We found that triggering vascular endothelial Pfn1 gene deletion, either in a widespread manner or locally in a kidney-restricted manner, suppresses tumor initiation and/or growth progression of pre-established kidney tumors in syngeneic mouse models. Loss of endothelial Pfn1 dramatically impacts the TME characterized by prominent suppression of tumor angiogenesis with massive tumor cell death and reduction of tumor-infiltrating immune cells. By performing cytokine array and Luminex assays of tumor lysates, we further identified Pfn1-dependent downregulation of several pro-angiogenic cell-secreted factors and pro-inflammatory cytokines that are major drivers of tumor progression in RCC including VEGF, endothelin-1, Cyr61, Macrophage Inflammatory Protein, G-CSF and IL6 in vivo. As a clinical correlate for mouse model data, we performed multiplexed quantitative immunohistochemistry analyses of human ccRCC specimens constructed on tissue microarray which also showed a positive correlation between Pfn1 expression (either in endothelial cells or tumor cells) and tumor infiltration of predominantly macrophages. Furthermore, through small molecule screening, we identified a novel inhibitor of the Pfn1-actin interaction (Pfn1i) and showed its ability to inhibit tumor angiogenesis and growth aggressiveness of tumor cells resembling various genetic landscapes of human ccRCC. Collectively, these findings establish endothelial Pfn1 as a critical regulator of TME and tumor progression in RCC, and provide a proof-of-concept for targeting Pfn1 as a potentially novel therapeutic strategy in RCC. Citation Format: David M. Gau, Abigail Allen, Andrew Daoud, Jessica Kunkel, Stefan Duensing, Partha Roy. Genetic disruption of vascular endothelial profilin-1 impacts tumor microenvironment supressing tumorigenicity of renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4584.

Abstract 4605: Chronic VEGF-C signaling exacerbates the progression of non-alcoholic steatohepatitis and hepatocarcinoma through endothelial VEGFR2 and VEGFR3

Abstract Obesity, aging, and metabolic diseases contribute to non-alcoholic fatty liver disease (NAFLD) that can progress to non-alcoholic steatohepatitis (NASH) due to ongoing inflammation and pathological angiogenesis. At present, NASH is a leading cause of cirrhosis, hepatocarcinoma cancer (HCC) and liver transplant within the United States. Pathological angiogenesis of liver sinusoidal endothelial cells (LSECs) is one of the hallmarks of liver injury that disrupt normal LSEC physiology. Therefore, strategies which target factors involved in LSEC dysregulation may improve the progression of hepatic diseases. Examination of a human tissue microarray identified increased levels of vascular endothelial growth factor-C (VEGF-C) in NASH livers compared with healthy individuals. Additionally, single nuclei RNA sequencing and immunofluorescent staining revealed that the receptors for VEGF-C, VEGFR2 and VEGFR3, are primarily expressed by the LSECs. Using murine models of NASH, we further investigated the role of VEGF-C during disease progression. Chronic over expression of VEGF-C by adeno-associated viral (AAV) infection in mice fed a western diet with carbon tetrachloride injections increased NASH progression compared with control AAV infected NASH mice. Interestingly, overexpression of VEGF-C C156S, a variant of VEGF-C which exclusively binds to VEGFR3, resulted in significantly increased hepatic steatosis, but only minimal fibrosis. To determine whether blockade of VEGF-C signaling would delay disease progression, we administered lenvatinib, a tyrosine kinase inhibitor which primarily blocks VEGFR2 and VEGFR3 at low doses, to NASH mice. We discovered that low dose lenvatinib resulted in significantly decreased amount of fibrosis, steatosis, and tumor formation. Analysis of vehicle treated and lenvatinib treated livers by single nuclei RNA sequencing uncovered significant changes in endothelial cell genes associated with extracellular interactions and inflammation, as well as hepatocyte genes involved in lipid synthesis and metabolism. Taken together, these findings indicate that chronic VEGF-C production in NASH plays a role in promoting liver fibrosis and steatosis as well as HCC development, and that blockade of the downstream receptors for VEGF-C, VEGFR2 and VEGFR3, may be a promising therapeutic strategy to mitigate disease severity. Citation Format: Seock-Won Youn, Jason W.-L. Eng, Bhairavi Swaminathan, Pamela Teneqexhi, Rahul Vadakath, Jan K. Kitajewski. Chronic VEGF-C signaling exacerbates the progression of non-alcoholic steatohepatitis and hepatocarcinoma through endothelial VEGFR2 and VEGFR3. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4605.

Endothelial nitric oxide synthase rs1799983 gene polymorphism is associated with the risk of developing intracranial aneurysm.

The intracranial aneurysm (IA) rupture is associated with a subarachnoid hemorrhage. One third of patients die, and one third remain depend for daily activities. Genetic factors are crucial in the formation and clinical evolution of IAs. Multiple loci have been associated with AIs, much of them implicating multiple pathways related to vascular endothelial maintenance and extracellular matrix integrity. Thus, the aim of our study was to characterize whether polymorphisms in genes implicated in the vascular endothelial maintenance could modify the risk of developing IAs. We have studied 176 patients with IA recruited in the Service of Neurosurgery at the University Hospital of Valladolid (Spain) and a control group if 150 sex-matched healthy subjects. Clinical variables were collected from each patient. We have analyzed VEGFA rs833061, VEGFR2 rs2071559, endothelin rs5370, endoglin rs3739817, and eNOS rs1799983 polymorphisms. Our results showed that allele T of the eNOS rs1799983 polymorphism is correlated with decreased risk of developing the disease; thus, allele G of the eNOS rs1799983 polymorphism increased the risk of developing IA. The association of eNOS rs1799983 polymorphism with the risk to suffer IA reinforces the hypothesis that genetic variants in eNOS gene could be crucial in the pathogenesis of IA.

Uncovering the Gene Regulatory Network of Endothelial Cells in Mouse Duchenne Muscular Dystrophy: Insights from Single-Nuclei RNA Sequencing Analysis.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by mutations in the dystrophin gene, which leads to heart and respiratory failure. Despite the critical impact of DMD on endothelial cells (ECs), there is limited understanding of its effect on the endothelial gene network. The aim of this study was to investigate the impact of DMD on the gene regulatory network of ECs. To gain insights into the role of the dystrophin muscular dystrophy gene (DMD) in ECs from Duchenne muscular dystrophy; the study utilized single-nuclei RNA sequencing (snRNA-seq) to evaluate the transcriptomic profile of ECs from skeletal muscles in DMD mutant mice (DMDmut) and wild-type control mice. The analysis showed that the DMD mutation resulted in the suppression of several genes, including SPTBN1 and the upregulation of multiple long noncoding RNAs (lncRNAs). GM48099, GM19951, and GM15564 were consistently upregulated in ECs and skeletal muscle cells from DMDmut, indicating that these dysregulated lncRNAs are conserved across different cell types. Gene ontology (GO) enrichment analysis revealed that the DMD mutation activated the following four pathways in ECs: fibrillary collagen trimer, banded collagen fibril, complex of collagen trimers, and purine nucleotide metabolism. The study also found that the metabolic pathway activity of ECs was altered. Oxidative phosphorylation (OXPHOS), fatty acid degradation, glycolysis, and pyruvate metabolism were decreased while purine metabolism, pyrimidine metabolism, and one carbon pool by folate were increased. Moreover, the study investigated the impact of the DMD mutation on ECs from skeletal muscles and found a significant decrease in their overall number, but no change in their proliferation. Overall, this study provides new insights into the gene regulatory program in ECs in DMD and highlights the importance of further research in this area.

Transcriptome analysis reveals tumor microenvironment changes in glioblastoma

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n= 322 test, n= 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

Smad4-mediated angiogenesis facilitates the beiging of white adipose tissue in mice

Beige adipocytes are thermogenic with high expression of uncoupling protein 1 in the white adipose tissue (WAT), accompanied by angiogenesis. Previous studies showed that Smad4 is important for angiogenesis. Here we studied whether endothelial Smad4-mediated angiogenesis is involved in WAT beiging. Inducible knockout of endothelial cell (EC) selective Smad4 (Smad4 iEC-KO) was achieved by using the Smad4 Floxp/floxp and Tie2 CreERT2 mice. Beige fat induction achieved by cold or adrenergic agonist, and angiogenesis were attenuated in WAT of Smad4 iEC-KO mice, with the less proliferation of ECs and adipogenic precursors. RNA sequencing of human ECs showed that Smad4 is involved in angiogenesis-related pathways. Knockdown of SMAD4 attenuated the upregulation of VEGFA, PDGFA, and angiogenesis invitro. Treatment of human ECs with palmitic acid-induced Smad1/5 phosphorylation and the upregulation of core endothelial genes. Our study shows that endothelial Smad4 is involved in WAT beiging through angiogenesis and the expansion of adipose precursors into beige adipocytes.

Biogenic copper oxide nanoparticles from Bacillus coagulans induced reactive oxygen species generation and apoptotic and anti-metastatic activities in breast cancer cells

The present study examined the anticancer capabilities of Bacillus coagulans supernatant-produced copper oxide nanoparticles (BC-CuONPs) on MCF-7 and SKBR3 cancer cells. The X-ray diffraction, ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray, dynamic light scattering, and zeta potential techniques were used to characterize BC-CuONPs. This study also investigated the cellular and molecular processes of NPs’ anti-proliferative and apoptotic properties on human breast cancer cells and compared them to the commercial pharmaceutical tamoxifen. The size of the spherical NP was from 5 to 47 nm with negative zeta potential. The MTT results showed the great cytotoxic effect of BC-CuONPs against breast cancer cells. The BC-CuONPs inhibited the growth of breast cancer cells in a time- and dose-dependent manner. The up-regulation of BCL2-associated X (BAX), cyclin dependent kinase inhibitor 1A (P21), Caspase 3 (CASP3), and Caspase 9 (CASP9), the down-regulation of BCL2 apoptosis regulator (BCL2), Annexin V-FITC/propidium iodide, and reactive oxygen species (ROS) generation results suggested that BC-CuONPs had a significant apoptotic impact when compared to the control. Scratch tests and vascular endothelial growth factor receptor gene (VEGF) down-regulation demonstrated that BC-CuONPs had anti-metastatic activity. The cell cycle analysis and down-regulation of Cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) revealed that cancer cells were arrested in the sub-G1 phase. Finally, the results showed that the secondary metabolites in the supernatant of Bacillus coagulans could form CuONPs, and biogenic BC-CuONPs showed anti-metastasis and anticancer properties on breast cancer cells while having less adverse effects on normal cells. Therefore, the synthesized CuONPs using B. coagulans supernatant can be shown as a potential candidate for a new therapeutic strategy in cancer management.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

Abstract TP181: Identification Of Hub Genes In Endothelial Cells Under Oscillatory Flow

Objective: Abnormal blood flow can influence gene expression of endothelial cells. Atherosclerotic plaques are more likely to form at the arterial bifurcation or the downstream of stenosis with low or oscillatory wall shear stress (WSS). However, the mechanism remains elusive. This study aims to explore the hub genes and the transcription factors (TF) regulating endothelial gene expression under oscillatory flow through bioinformatics. Methods: Microarray expression profile (GSE16706) was obtained from the Gene Expression Omnibus database to explore differentially expressed genes (DEGs) of human umbilical vein endothelial cells (HUVECs) under oscillatory flow (time-average 1 dyne/cm2; range: -11 - +11 dynes/cm2; 1 Hz). Enrichment analysis was conducted to infer the function of DEGs. Protein-protein interaction network was also plotted to further explore hub genes. TF for hub genes were predicted through NetworkAnalyst. Results: 303 DEGs, with 219 up-regulated genes and 84 down-regulated genes, were identified by comparing gene expression of HUVECs under oscillatory flow or static condition. 4 hub genes (CCNA2, CDC45, BUB1B, KIF20A) related to cell cycle were verified in GSE83476. ELF1 which could regulate all these 4 hub genes was selected as a key TF. Conclusion: We found 4 hub genes and 1 TF associated with oscillatory flow treated HUVECs, which might also be involved in oscillatory flow induced atherosclerosis.

Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during postnatal growth

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

Psychological measures of stress and biomarkers of inflammation, aging, and endothelial dysfunction in breast cancer survivors on aromatase inhibitors

The use of aromatase inhibitors (AIs) is associated with higher rates of cardiovascular events and lower endothelial function in breast cancer survivors. Psychosocial stress is associated with higher levels of inflammatory and aging markers, and lower endothelial function in otherwise healthy subjects. These associations among breast cancer survivors on AIs are not well defined. A cross-sectional study of 30 breast cancer survivors on AIs was performed to assess the associations between self-reported scores of psychosocial measures of depression, anxiety, and stress assessed by validated questionnaires with markers of inflammation (CRP; IL-6; IL-18), aging (p16INK4a), and endothelial function (ICAM-1, EndoPAT ratio). Significant positive correlations were observed between psychosocial measures and inflammatory markers including CRP, IL-6, and ICAM-1. However, no psychosocial scores were related to endothelial function or gene expression of the aging biomarker p16INK4a. Overall, survivors had endothelial dysfunction with reduced EndoPAT ratios. Psychosocial stress is associated with greater inflammation in breast cancer survivors on AIs, corroborating previous studies in cancer-free populations. The lack of association between psychosocial stress and either endothelial function or aging biomarkers could be due to the already low endothelial function and accelerated aging in our cohort of breast cancer survivors on AIs, though our small sample size limits conclusions. Further work in a larger and more diverse cohort of patients is needed to further understand the relationships among inflammation, aging and endothelial function in breast cancer survivors.

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

A C2H2 zinc finger transcription factor of the whitefly Bemisia tabaci interacts with the capsid proteins of begomoviruses and inhibits virus retention.

Begomoviruses are a group of ssDNA viruses exclusively transmitted by the whitefly Bemisia tabaci and constrain vegetable production in the old and new worlds. Although multiple molecular determinants governing the transmission of begomoviruses by whiteflies have been unravelled, factors critical for transmission majorly remain unknown. In this study, a whitefly C2H2 zinc finger (ZF) protein, 100% identical to the vascular endothelial ZF-like gene (vezf) protein was confirmed to interact with the CP of both old- and new-world begomoviruses. This was achieved by a yeast two-hybrid (Y2H) system screening of a whitefly cDNA library using capsid protein (CP) of TYLCV as a bait. In silico annotation of vezf protein revealed that it contains a N-terminal ZF-associated domain (ZAD) alongside multiple C2H2 ZF domains on the C-terminal end. ZAD-ZF proteins form the most abundant class of transcription factors within insects. Herein, we validated the interaction of vezf with four diverse begomoviruses and its functional role in begomovirus transmission. Silencing of the vezf gene of B. tabaci led to increased retention of three diverse begomoviruses tested. Vezf is the first insect transcription factor identified to interact with plant viruses and can be crucial to understand the possible mechanisms by which plant viruses modulate transcription of their insect vectors during transmission.

Sphingosine-1-phosphate related signalling pathways manipulating virus replication.

Viruses can create a unique cellular environment that facilitates replication and transmission. Sphingosine kinases (SphKs) produce sphingosine-1-phosphate (S1P), a bioactive sphingolipid molecule that performs both physiological and pathological effects primarily by activating a subgroup of the endothelial differentiation gene family of G-protein coupled cell surface receptors known as S1P receptors (S1PR1-5). A growing body of evidence indicates that the SphK/S1P axis is crucial for regulating cellular activities in virus infections like respiratory viruses, enteroviruses, hepatitis viruses, herpes viruses, and arboviruses replicate. Depending on the type of virus, pro- or anti-viral activities of the SphK/S1P axis sometimes rely on the host immune system and sometimes directly through intracellular signalling pathways or cell proliferation. Recent research has shown novel roles of S1P and SphK in viral replication. Sphingosine kinase isoforms (SphK1 and SphK2) levels can be manipulated by several viruses to promote the effects that are expected. Regulation of cellular signalling pathways plays a significant role in the mechanism. The purpose of this review is to provide insight of the characters played by the SphK/S1P axis throughout diverse viral infection processes. We then assess potential therapeutic methods that are based on S1P signalling and metabolism during viral infections.

Эндотелиальная дисфункция при привычном невынашивании беременности: генетические факторы и факторы внешней среды

Objective. To examine the association between the carriage of endothelial dysfunction-related gene polymorphisms and recurrent pregnancy loss (RPL). Patients and methods. This case-control study included 87 patients who were divided into two groups. The study group consisted of 65 patients with a history of two or more consecutive pregnancy losses (2 to 11 losses). Primary RPL was detected in 20 patients, secondary in 26, and tertiary in 19. The control group consisted of 22 women with a history of three or more full-term births without episodes of miscarriage. The frequency of polymorphisms of the VEGFA -634G>C and IL6 -174G>C genes was determined by a ready-to-use real-time polymerase chain reaction (RT-PCR) test kits. Results. Carrying the VEGFA -634G>C GC genotype increases the risk of RPL 4-fold (p = 0.0072) and the IL6 -174G>C GC genotype – 3-fold (p = 0.028). Analysis of the impact of combinations of unfavorable genotypes showed that 65.0% of patients with primary RPL, 61.5% with secondary RPL, 63.2% with tertiary RPL, and 9% of healthy controls had a combination of GC VEGFA -634G>C and CC IL6 -174G>C. One of the key environmental factors determining the realization of susceptibility genes is age: the maximum age at the time of the first intended pregnancy was observed in patients with primary RPL (32.6 years vs. 25.5 years in the control group; p < 0.05), with 9% of healthy controls having a combination of two genotypes associated with pregnancy loss. That is, successful pregnancy at a younger age is possible even when carrying multiple risk alleles. The birth interval length is also important for successful pregnancy: a short inter-pregnancy interval allows to carry out the reproductive function during the most favorable period of the reproductive system before the influence of genotypes predisposing to RPL begins. Other environmental factors leading to the realization of carriage of RPL risk alleles include a history of intrauterine interventions and sexually transmitted infections. Conclusion. Study of the VEGFA -634G>C and IL6 -174G>C gene polymorphisms may serve as an effective method to predict RPL. For patients carrying these unfavorable gene polymorphisms, it is especially important to perform the reproductive function in time, i.e., before the realization of environmental factors. Key words: recurrent pregnancy loss, VEGF, endothelial dysfunction, susceptibility genes

PS-C30-3: FEATURES OF THE C-786T NOS3 GENE POLYMORPHISM IN PREGNANT WOMEN WITH PREECLAMPSIA

Relevance: Preeclampsia is the multi cause illness. One of the genetic risk factors for the development of preeclampsia may be a polymorphism of the endothelial system genes. Objective: to study the C-786T polymorphism of the NOS3 gene in patients with preeclampsia. Materials and methods: The study group consisted of 140 pregnant women, of which the main group included 72 patients with preeclampsia, and 68 apparently healthy individuals (control group). The study was conducted in the Tashkent city maternity complex 6. The age of the pregnant women ranged from 19 to 42 years. The material for the study was DNA samples from a patient with a clinically diagnosed preeclampsia. Statistical processing of the results was carried out using statistical programs “EpiCalc 2000 Version 1.02“. Results: The proportion of wild C allele in the subgroup of patients was statistically significantly lower compared to the control (68.6% versus 79.9%, respectively; (p = 0.04; RR = 0.86; OR = 0.55; 95%). On the contrary, the frequency of the unfavorable T allele among patients with severe preeclampsia was significantly higher compared to controls - 31.4% and 20.1%, respectively (p = 0.04). At the same time, the calculated odds ratio for detecting this allele was OR = 1.8, and the relative risk of developing diseases was RR = 1.17 (p = 0.04). There was a trend towards an increase in the heterozygous C/T genotype in the group of patients with severe preeclampsia, where it was detected with a frequency of 39.5% compared to 27.1% in the control group (p = 0.15; RR = 1.46; OR = 1.76). The functionally unfavorable T/T genotype was insignificantly more common among patients with severe preeclampsia compared to the control group 11.6% and 6.5%, respectively (p = 0.30; RR = 1.78; OR = 1.88;). A significant relationship was found between the risk of developing preeclampsia and the carriage of a rare allelic variant of the C-786T polymorphism of the NOS3 gene, associated with a decrease in the synthesis of the NO-synthetase enzyme. Conclusions: Thus, the results of our study allow us to conclude that unfavorable genotypic variants of the C-786T polymorphism of the NOS3 gene (associated with vascular endothelial dysfunction) contribute significantly to the mechanism of preeclampsia development.

HAND1 knockdown disrupts trophoblast global geneexpression.

Congenital heart disease (CHD) affects nearly 1% of births annually, and CHD pregnancies carry increased risk of developing pathologies of abnormal placentation. We previously reported significant developmental impacts of disrupting Hand1, a gene associated with CHD, expression in placenta trophoblast and endothelial cells in multiple mouse models. In this study, we aimed to build upon this knowledge and characterize the mechanistic impacts of disrupting HAND1 on human placenta trophoblast and vascular endothelial cell gene expression. HAND1 gene expression was silenced in BeWo cells, a choriocarcinoma model of human cytotrophoblasts, (n=3-9 passages) and isolated human placental microvascular endothelial cells (HPMVEC; n=3 passages), with HAND1 siRNA for 96 h. Cells were harvested, mRNA isolated and RNA sequencing performed using the Illumina NextSeq 550 platform. Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR packages. Statistical significance was determined using a log2 fold change of >1.0 or < -1.0 and unadjusted p-value ≤0.05. Panther DB was used for overrepresentation analysis, and String DB for protein association network analysis. There was downregulation of 664 genes, and upregulation of 59 genes in BeWo cells with direct HAND1 knockdown. Overrepresentation analysis identified disruption to pathways including cell differentiation, localization, and cell projection organization. In contrast, only seven genes were changed with direct HAND1 knockdown in HPMVECs. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not endothelial cells. This data provides further evidence that future studies on genetic perturbations in CHDs should consider the extra-embryonic tissue in addition to the fetal heart.