Apelin Signaling Research Articles

Interaction Between Functionally Activate Endometrial Microbiota and Host Gene Regulation in Endometrial Cancer.

Objective: In this study, we mainly explored two questions: Which microorganisms were functionally active in the endometrium of patients with endometrial cancer (EC)? What kind of response did the human host respond to functionally active microorganisms?Methods: Nine endometrial cancer patients and eight normal subjects were included in this study. HMP Unified Metabolic Analysis Network 3 (HUMAnN3) was used to obtain functional information of microorganisms. In addition, metaCyc-based GSEA functional enrichment analysis was used to obtain information on the metabolic pathways of the human host. At the same time, the O2PLS model and Spearman correlation analysis were used to analyze the microorganisms–host interaction.Results: With the novel metatranscriptome analysis pipeline, we described the composition of more than 5,000 functionally active microorganisms and analyzed the difference in microorganisms between the EC and the normal group. Our research found that these microorganisms were involved in part of the metabolic process of endometrial cancer, such as 6-sulfo-sialyl Lewis x epitope, N-acetyl-beta-glucosaminyl. In addition, the host–microbiota crosstalk of EC endometrium also included many biological processes, mainly functions related to tumor migration and the Apelin signaling pathway.Conclusion: The functionally active microorganisms in the EC endometrium played an essential role in the occurrence and migration of tumors. This meant that functionally active microorganisms could not be ignored in the treatment of endometrial cancer. This study helped to better understand the possible role of endometrial functional, active microorganisms in the occurrence and development of EC in patients with endometrial cancer and provided new information for new attempts to treat EC.

Identification of tRNA-derived Fragments and Their Potential Roles in Atherosclerosis

Atherosclerosis (AS), a chronic inflammatory disease, is the basis of cardiovascular disease (CVD). Although the treatment has been greatly improved, AS still imposes a large burden on human health and the medical system, and we still need to further study its pathogenesis. As a novel biomolecule, transfer RNA-derived fragments (tRFs) play a key role in the progression of various disease. However, whether tRFs contribute to atherosclerosis pathogenesis remains unexplored. With deep sequencing technology, the change of tRFs expression profiles in patients with AS compared to healthy control group was identified. The accuracy of the sequencing data was validated using RT qPCR. Subsequently, we predicted the potential target genes of tRFs by online miRNA target prediction algorithms. The potential functions of tRFs were evaluated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. There were 13 tRFs differentially expressed between patients with AS and healthy controls, of which 2 were up-regulated and 11 were down-regulated. Validation by RT-qPCR analysis confirmed the sequencing results, and tRF-Gly-GCC-009 was highly up-regulated in the AS group based on the results of sequencing which was confirmed by RT-qPCR analysis. Furthermore, GO enrichment and KEGG pathway analyses indicated that 10 signaling pathways were related to tRF-Gly-GCC-009. These pathways might be physiopathological fundamentals of AS, mainly involving in Apelin signaling, Notch signaling and calcium signaling. The results of our study provide important novel insight into the underlying pathogenesis and demonstrate that tRFs might be potential biomarkers and therapeutic targets for AS in the future.

Receptor Tyrosine Kinase Inhibitor Sunitinib as Novel Immunotherapy to Inhibit Myeloid-Derived Suppressor Cells for Treatment of Endometriosis.

Endometriosis is a common, benign, and hormone-dependent gynaecological disorder that displays altered immunoinflammatory profiles. Myeloid-derived suppressor cells (MDSCs) suppressed immunosurveillance in endometriosis in human and mouse model. Receptor tyrosine kinase inhibitor Sunitinib can induce MDSC apoptosis and suppress the progression of cancer. However, the effects of Sunitinib on MDSCs in endometriosis and the underlying mechanism are not clear. In this study, we employed an animal study of the endometriosis model in mice for treatment of Sunitinib. After syngeneic endometrium transplantation and treatment, endometriotic lesion volume, weight, and histology were compared. Peritoneal fluid, peripheral blood, and bone marrow MDSC subsets and their molecular signaling were monitored by flow cytometry. Peritoneal cytokines were assayed by ELISA. The gene expression profiles of isolated CD11b+Ly6G+Ly6Clo cells were studied by RNA sequencing. We found that Sunitinib significantly decreased the endometriotic lesion size and weight after 1 and 3 weeks, and decreased p-STAT3 activation in MDSCs after 1 week of treatment. In the first week, Sunitinib specifically increased the G-MDSC population in peritoneal fluid but the isolated CD11b+Ly6G+Ly6Clo MDSCs after Sunitinib treatment were presented as mature polynuclear MDSCs, while the control group had immature mononuclear MDSCs. Importantly, we found Sunitinib differentially suppressed gene expressions of immunosuppressive function and differentiation in peritoneal G-MDSCs. Apelin signaling pathway associated genes and inflammation related genes were upregulated, and amino acid metabolism regulator genes were downregulated in bone marrow G-MDSCs. For endometriotic lesions, the PPARG gene governing glucose metabolism and fatty acid storage, which is important for the development of endometriosis was upregulated. In conclusion, Sunitinib inhibited endometriotic lesions, by promoting peritoneal fluid MDSCs maturation and inhibiting the immunosuppressive function. These findings suggest that Sunitinib changed the immune microenvironment and inhibited the development of endometriosis, which has potential therapeutic effects as novel immunotherapy to promote MDSCs maturation, differentiation, and metabolism for the treatment of endometriosis.

Different combinations of high-frequency rTMS and cognitive training improve the cognitive function of cerebral ischemic rats

Poststroke cognitive impairment (PSCI) occurs frequently after stroke, but lacks effective treatments. Previous studies have revealed that high-frequency repetitive transcranial magnetic stimulation (rTMS) has a beneficial effect on PSCI and is often used with other cognitive training methods to improve its effect. This study aimed to evaluate the effect of different combinations of rTMS and cognitive training (rTMS-COG) on PSCI and identify the optimal combination protocol. A cerebral infarction rat model was established by transient middle cerebral artery occlusion (tMCAO). The Morris water maze test was conducted to assess the cognitive function of rats. RNA sequencing and bioinformatics analysis were employed to study the underlying mechanisms. rTMS, COG and rTMS-COG all had beneficial effects on PSCI, while cognitive training immediately after rTMS (rTMS-COG0h) achieved a better effect than cognitive training 1 h and 4 h after rTMS, rTMS and COG. We identified 179 differentially expressed genes (DEGs), including 24 upregulated and 155 downregulated genes, between the rTMS-COG0h and rTMS groups. GO analysis revealed that the major categories associated with the DEGs were antigen procession and presentation, regulation of protein phosphorylation and axoneme assembly. KEGG analysis showed that the DEGs were enriched in processes related to phagosome, circadian entrainment, dopaminergic synapse, apelin signaling pathway, long-term depression, neuroactive ligand-receptor interaction, axon guidance and glucagon signaling pathway. PPI analysis identified Calb2, Rsph1, Ccdc114, Acta2, Ttll9, Dnah1, Dlx2, Dlx1, Ccdc40 and Ccdc113 as related genes. These findings prompt exploration of the potential mechanisms and key genes involved in the effect of rTMS-COG0h on PSCI.

The biological regulatory activities of Flammulina velutipes polysaccharide in mice intestinal microbiota, immune repertoire and heart transcriptome

The effects of a novel Flammulina velutipes polysaccharide (FVP) on intestinal microbiota, immune repertoire and heart transcriptome were investigated in this study. The results showed that FVP treatment could effectively regulate the abundance of colonic microbiota. And FVP exhibited obvious immunoregulatory effect by influencing V gene and J gene fragments usage on TCRα chain. The usage frequency of TRBV1, TRBJ1-6 and TRBJ1-5 were significantly altered, and 41 V-J pairs were identified with obvious difference after FVP treatment. Furthermore, the mRNA of mice heart was analyzed by transcriptome assay. Total 525 genes and 1587 mRNA were significantly changed after FVP treatment. KEGG annotation indicated that the up-regulated mRNA was enriched in 17 pathways including adherens junction, mTOR signaling pathway, insulin signaling pathway, mitophagy, tight junction, PPAR signaling pathway and TNF signaling pathway, etc. Meanwhile, the down-regulated mRNA was gathered in AMPK signaling pathway, metabolism of xenobiotics by cytochrome P450, apelin signaling pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, insulin signaling pathway, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, Fc gamma R-mediated phagocytosis, etc. The great potential exhibited by FVP could make it an ideal candidate as complementary medicine or functional food for promotion of health.

Characterization of the proteome and metabolome of human liver sinusoidal endothelial-like cells derived from induced pluripotent stem cells

The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

Small RNA Sequencing of Aqueous Humor and Plasma in Patients With Primary Open-Angle Glaucoma.

PurposeIdentify differentially expressed microRNAs (miRNAs) in aqueous humor (AH) and blood of primary open-angle glaucoma (POAG) patients by using small RNA sequencing. These may provide insight into POAG pathophysiology or serve as diagnostic biomarker.MethodsAH and plasma of nine POAG patients and 10 cataract control patients were small RNA sequenced on Illumina NovaSeq 6000. Identification of gene transcripts targeted by differentially expressed miRNAs was done with miRWalk and MirPath. These targets were used for pathway analysis and Gene Ontology enrichment. Diagnostic potential was evaluated by receiver operating characteristics analysis.ResultsWe identified 715 miRNAs in plasma and 62 miRNAs in AH. Plasma miRNA profile did not differ between POAG and control. In contrast, in AH, seven miRNAs were differentially expressed. Hsa-miR-30a-3p, hsa-miR-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were upregulated, whereas hsa-miR-92a-3p, hsa-miR-451a, and hsa-miR-486-5p were downregulated in POAG. Compared to previous studies, hsa-mir-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were reported previously, strengthening their involvement in POAG whereas hsa-miR-30a-3p, hsa-miR-92a-3p, and hsa-miR-486-5p are implicated in POAG for the first time. Identified gene transcripts were involved in several pathways, some implicated in glaucoma before (e.g., TGF-β and neurotrophin signaling), whereas others are new (e.g., prolactin and apelin signaling). In respect to diagnostics, AH concentration of hsa-mir-143-3p had an area under the curve (AUC) of 0.889. Combined with hsa-miR-221-3p, AUC improved to 0.96.ConclusionsSmall RNA sequencing identified seven differentially expressed miRNAs in AH of POAG patients. The differentially expressed miRNAs may be useful as POAG biomarkers or could become targets for new therapeutic strategies.

Hypothalamic and ovarian transcriptome profiling reveals potential candidate genes in low and high egg production of white Muscovy ducks (Cairina moschata)

In China, the low egg production rate is a major challenge to Muscovy duck farmers. Hypothalamus and ovary play essential role in egg production of birds. However, there are little or no reports from these tissues to identify potential candidate genes responsible for egg production in White Muscovy ducks. A total of 1,537 laying ducks were raised; the egg production traits which include age at first egg (days), number of eggs at 300 d, and number of eggs at 59 wk were recorded. Moreover, 4 lowest (LP) and 4 highest producing (HP) were selected at 59 wk of age, respectively. To understand the mechanism of egg laying regulation, we sequenced the hypothalamus and ovary transcriptome profiles in LP and HP using RNA-Seq. The results showed that the number of eggs at 300 d and number of eggs at 59 wk in the HP were significantly more (P < 0.001) than the LP ducks. In total, 106.98G clean bases were generated from 16 libraries with an average of 6.68G clean bases for each library. Further analysis showed 569 and 2,259 differentially expressed genes (DEGs) were identified in the hypothalamus and ovary between LP and HP, respectively. The KEGG pathway enrichment analysis revealed 114 and 139 pathways in the hypothalamus and ovary, respectively which includes Calcium signaling pathway, ECM-receptor interaction, Focal adhesion, MAPK signaling pathway, Apoptosis and Apelin signaling pathways that are involved in egg production. Based on the GO terms and KEGG pathways results, 10 potential candidate genes (P2RX1, LPAR2, ADORA1, FN1, AKT3, ADCY5, ADCY8, MAP3K8, PXN, and PTTG1) were identified to be responsible for egg production. Further, protein-protein interaction was analyzed to show the relationship between these candidate genes. Therefore, this study provides useful information on transcriptome of hypothalamus and ovary of LP and HP Muscovy ducks.

10-LB: The Apelinergic System Improves Angiogenesis and Blood Flow Reperfusion in Diabetic Mouse Model of Lower Limb Ischemia

Introduction: Peripheral vascular disease is a major risk factor for lower-extremity amputation in patients with diabetes due to an abnormal angiogenic response. The apelinergic system is known to improve cardiac function after ischemia by promoting angiogenesis. The objective: is to study the effects of Apelin-13 (Ape-13) and Elabela (Ela) on blood flow (BF) reperfusion following limb ischemia in diabetic mice as well as on endothelial cell (EC) function. Method: Primary EC were exposed to normal (5.6 mM; NG) or high (25 mM; HG) glucose concentrations for 24h and hypoxia (1% O2) for the last 16h. EC were stimulated or not with Ape-13 or Ela. Proliferation and migration assays were performed and apelin signaling pathway was evaluated by immunoblot and qPCR. In vivo, nondiabetic (NDM) and diabetic (DM) underwent femoral artery ligation to induce lower limb ischemia. DM mice received Ape-13 or Ela by subcutaneous osmotic pump. BF reperfusion was measured for 4 weeks post-surgery and exercise willingness was assessed in individual cages with voluntary wheel. The ischemic abductor muscles were collected for protein, genes and vascular density analyses. Results: Inhibitory effects of HG exposure on EC migration and proliferation were prevented with Ape-13 and Ela treatment under hypoxia condition through activation of Akt, AMPK and Rock2. In vivo, our results indicated that BF reperfusion was recovered at 75% in NDM as compared to 37% in DM whereas Ape-13 and Ela treatment in DM mice improved BF reperfusion by 65% and 69%, respectively. Similar results were observed using voluntary wheel as Ape-13 and Ela enhanced distance run as compared to untreated DM mice. Finally, the expression of proangiogenic factors (VEGF-A, PDGF-B) was increased in the ischemic muscle of DM+Ape-13 or Ela as compared to DM mice. Conclusion: We believe that stimulation of apelinergic system improved BF recovery in DM by increasing proangiogenic pathways and EC migration and proliferation. Disclosure S. Robillard: None. P. Geraldes: None. Funding Canadian Institutes of Health Research (PJT159627)

Apelin promotes blood and lymph vessel formation and the growth of melanoma lung metastasis

Apelin, a ligand of the APJ receptor, is overexpressed in several human cancers and plays an important role in tumor angiogenesis and growth in various experimental systems. We investigated the role of apelin signaling in the malignant behavior of cutaneous melanoma. Murine B16 and human A375 melanoma cell lines were stably transfected with apelin encoding or control vectors. Apelin overexpression significantly increased melanoma cell migration and invasion in vitro, but it had no impact on its proliferation. In our in vivo experiments, apelin significantly increased the number and size of lung metastases of murine melanoma cells. Melanoma cell proliferation rates and lymph and blood microvessel densities were significantly higher in the apelin-overexpressing pulmonary metastases. APJ inhibition by the competitive APJ antagonist MM54 significantly attenuated the in vivo pro-tumorigenic effects of apelin. Additionally, we detected significantly elevated circulating apelin and VEGF levels in patients with melanoma compared to healthy controls. Our results show that apelin promotes blood and lymphatic vascularization and the growth of pulmonary metastases of skin melanoma. Further studies are warranted to validate apelin signaling as a new potential therapeutic target in this malignancy.

Abstract 16473: Loss of the Apelin-Apelin Receptor Signaling Axis Worsens Hemodynamic Alterations and Right Ventricular (rv) Adaptation and Abrogates 17ß-Estradiol (E2)-Mediated Protection in Experimental Pulmonary Arterial Hypertension (PAH)

Introduction: Women are more likely to develop PAH but exhibit more favorable hemodynamics and better survival than men. These improved outcomes have been linked to protective effects of E2. We recently linked RV-protective effects of E2 to regulation of apelin signaling. However, the role of apelin and its receptor APJ in RV failure is unknown. Hypothesis: RV cardiomyocytes (RVCM)-derived apelin exerts beneficial effects on RV endothelial cells (RVECs). Apelin-APJ signaling is necessary for E2’s RV-protective effects. Methods: Apelin and APJ abundance were quantified (Western blot) and immunolocalized in RVs from PAH patients. In vivo , RV failure was induced in male Sprague-Dawley rats by pulmonary artery banding (PAB). A subset of PAB rats were treated with E2 ± APJ inhibitor ML221. Hemodynamics, RV function and molecular changes were assessed. To study the role of RVCM apelin, conditioned media was collected from RVCMs after apelin knockdown and then added to RVECs. In addition, conditioned media from untreated RVCMs was added to RVECs pretreated with ML221. Lastly, RVECs were treated with E2 ± siApelin or ML221. p&lt;0.05 was considered statistically significant. Results: In PAH RVs, apelin and APJ localized to RVCMs and RVECs and were decreased vs controls (p&lt;0.05). In PAB+E2+ML221 rats, inhibition of apelin signaling resulted in more RV hypertrophy and decreased RV function (p&lt;0.05 vs sham or PAB+E2). Conditioned media from RVCMs+siApelin reduced RVEC eNOS phosphorylation, transwell migration and ring formation (p&lt;0.05 vs scrambled control conditioned media). Concomitantly, treatment of RVECs with ML221 blocked transwell migration and ring formation induced by RVCM conditioned media (p&lt;0.05 vs RVEC+veh control). Lastly, treatment of RVECs with siApelin or ML221 abrogated stimulatory effects of E2 on angiogenic function (p&lt;0.05). Conclusions: Apelin and APJ are decreased in PAH-RVs and loss of apelin-APJ signaling results in more severe RV failure and abrogates E2-mediated RV protection. Apelin is secreted by RVCMs and exerts a beneficial paracrine effect on RVECs, where it is required for E2 to stimulate angiogenesis. Apelin-APJ signaling may represent a novel and therapeutically targetable, cell- and sex-specific signaling axis in the RV.

Abstract 17259: Identification of Biological Pathways of Candidate Atrial Fibrillation Risk Genes Through the Use of Weighted Gene Coexpression Network Analysis

Introduction: Over 135 genetic loci have been linked to atrial fibrillation (AF), yet the biological pathways of AF pathophysiology remain elusive. Weighted gene coexpression network analysis (WGCNA) constructs gene modules within a network based on correlations in gene expression, and identifies mechanisms related to AF risk. Objective: To identify biological pathways of candidate AF risk genes that will advance our understanding of AF mechanisms. Methods: RNA-sequencing was performed on left atrial appendage tissue from 265 patients. RNA-seq data were adjusted for differences in AF rhythm state and other known AF risk factors. Correlations from adjusted data were further adjusted for latent factors then spatial quantile normalized to correct for mean-variance bias. WGCNA was applied to the resulting adjusted and normalized gene-gene correlations to identify gene modules. Ingenuity Pathway Analysis and gene set over representation analysis (GSOR) were applied to each module. Results: WGCNA identified 63 modules from 17,434 genes; 47 of these contained at least one candidate AF risk gene. AF risk genes were overrepresented in 7 modules (Table 1). Notable top pathways of AF overrepresented modules include apelin signaling, heme metabolism, intracellular ion homeostasis, and the unfolded protein response. These are known to be involved in calcium signaling, iron homeostasis, glucose regulation, heat shock response, and protein ubiquitination during states of high energy demand and stress. These pathways coincide with larger cellular processes of myocyte remodeling, apoptosis, and cell survival, which were also prominent. Conclusions: Biological pathways identified through WGCNA and GSOR suggest that sustained increases in energy demand during AF promotes stress-induced cellular remodeling. Changes in calcium signaling, iron homeostasis, the unfolded protein response and glucose regulation are likely primary mechanisms of AF pathophysiology.

MiR-15b-5p promotes expression of proinflammatory cytokines in human placenta by inhibiting Apelin signaling pathway

IntroductionThe timing of parturition at end of human gestation may be controlled by fetal signals. The signaling molecules contributing to activation of human labor may be mediated by fetal exosomes. In this study, we focused on investigation of the role of microRNAs (miRNAs) derived from fetal exosomes in the regulation of human placental gene expression. MethodsUsing immunofluorescent labeling, array-based miRNA profiling assay, target prediction analysis, and conducting a variety of biochemical approaches including miRNA mimics, dual-luciferase, siRNA-mediated gene silencing, and immunohistochemical staining assay in primary trophoblast culture and formalin-fixed paraffin-embedded placental tissues, we examined whether fetal exosomal miRNAs can stimulate expression of proinflammatory mediators in human placenta. ResultsWe showed placental uptake of exosomes derived from the umbilical artery, and found that 9 fetal exosomal miRNAs: let-7i-5p, miR-185-5p, miR-15b-5p, miR-376c-3p, miR-548d-5p, miR-92b-3p, miR-16-5p, and miR-1301-3p were significantly increased in placentas of women delivering following term labor compared to those delivering by Cesarean section in the late preterm period. Target prediction analysis identified miR-15b-5p of particular interest, because one of its predicted targets is Apelin, a potent inhibitor of proinflammatory mediators. We further found that miR-15b-5p repressed Apelin protein levels and activated pro-labor hormones and cytokines including IL-1, IL-6, IL-8, and TNF-α. DiscussionThese data suggest a potential fetal-to-placental signal that could play a role in the length of human gestation and onset of human labor.

Kynurenine-PARP 1 link mediated by microRNA 210 may be dysregulated in pulmonary hypertension

Abstract Background The aims of the present study were to determine the alterations in mRNA and miR expressions and their role in signaling pathways, to correlate their levels with the severity of PH, and to investigate the relationship between those alterations and serum levels of apelin, kynurenine, and endocan in PH. Methods The study included 32 treatment-naive patients with precapillary PH and 55 controls. All subjects underwent RHC. Total RNA was isolated and cDNAs for mRNA and miR were synthesized. mRNA expressions of HIF-1α, HIF-2α, STAT 3, FGF-2, FGFR-1, PARP 1 and miR expressions of miR-210, miR-130a, miR-424, miR-204, and miR-223 were determined. Concentrations of kynurenine, apelin, and endocan were analyzed. Results HIF-2α, STAT 3, and FGF-2 were increased; miR-210 and miR-130a were increased; miR-223 and miR-204 were decreased. Apelin and kynurenine concentrations were decreased. There were positive correlations: HIF-2α-miR-424: r=0.474, p=0.011; APLN-miR-424: r=0.385, p=0.030; kynurenine-miR-210: r=0.551, p=0.004; STAT 3- PVR: r=0.478, p=0.006; miR-210- RAP: r=0.536, p=0.07; kynurenine-RAP: r=0.409, p=0.022. There were negative correlations: PARP 1-miR-210: r=−0.561, p=0.007; PARP 1- RAP: r=−0.424, p=0.27. miR-130a (OR= 1.257, p=0.016) and APLN (OR= 0.223, p=0.004) were independent risk factors for PH. Conclusions We report: 1) novel relationship between the kynurenine and PARP 1 signaling pathways that could be mediated by miR-210, 2) relationship between the apelin and HIF-2α signaling pathways that could be mediated by miR-424, 3) reduced levels of apelin and elevated levels of miR-130a are associated with PH, 4) elevated levels of STAT 3, miR-210, and kynurenine, and reduced levels of PARP 1 correlate with more severe hemodynamics. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Pamukkale University Scientific Research Projects Coordination Unit

Angiotensin AT1 receptor antagonism by losartan stimulates adipocyte browning via induction of apelin

Adipocyte browning appears to be a potential therapeutic strategy to combat obesity and related metabolic disorders. Recent studies have shown that apelin, an adipokine, stimulates adipocyte browning and has negative cross-talk with angiotensin II receptor type 1 (AT1 receptor) signaling. Here, we report that losartan, a selective AT1 receptor antagonist, induces browning, as evidenced by an increase in browning marker expression, mitochondrial biogenesis, and oxygen consumption in murine adipocytes. In parallel, losartan up-regulated apelin expression, concomitant with increased phosphorylation of protein kinase B and AMP-activated protein kinase. However, the siRNA-mediated knockdown of apelin expression attenuated losartan-induced browning. Angiotensin II cotreatment also inhibited losartan-induced browning, suggesting that AT1 receptor antagonism-induced activation of apelin signaling may be responsible for adipocyte browning induced by losartan. The in vivo browning effects of losartan were confirmed using both C57BL/6J and ob/ob mice. Furthermore, in vivo apelin knockdown by adeno-associated virus carrying-apelin shRNA significantly inhibited losartan-induced adipocyte browning. In summary, these data suggested that AT1 receptor antagonism by losartan promotes the browning of white adipocytes via the induction of apelin expression. Therefore, apelin modulation may be an effective strategy for the treatment of obesity and its related metabolic disorders.

Apelin signaling drives vascular endothelial cells toward a pro-angiogenic state.

To form new blood vessels (angiogenesis), endothelial cells (ECs) must be activated and acquire highly migratory and proliferative phenotypes. However, the molecular mechanisms that govern these processes are incompletely understood. Here, we show that Apelin signaling functions to drive ECs into such an angiogenic state. Zebrafish lacking Apelin signaling exhibit defects in endothelial tip cell morphology and sprouting. Using transplantation experiments, we find that in mosaic vessels, wild-type ECs leave the dorsal aorta (DA) and form new vessels while neighboring ECs defective in Apelin signaling remain in the DA. Mechanistically, Apelin signaling enhances glycolytic activity in ECs at least in part by increasing levels of the growth-promoting transcription factor c-Myc. Moreover, APELIN expression is regulated by Notch signaling in human ECs, and its function is required for the hypersprouting phenotype in Delta-like 4 (Dll4) knockdown zebrafish embryos. These data provide new insights into fundamental principles of blood vessel formation and Apelin signaling, enabling a better understanding of vascular growth in health and disease.

Network analysis and experimental verification of Qingyan Formula in treatment of perimenopausal anxiety disorder

This paper aims to explore the potential of Qingyan Formula(QYF) in the treatment of perimenopausal anxiety disorder(PAD) by network pharmacological method, and verify the pharmacodynamics and mechanism by using animal experiments. First, Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP) was used to retrieve the active components of QYF. Then the potential drug targets were screened with use of Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine(TCMIP) v2.0 and Swiss Target Prediction database. Targets related to PAD were retrieved and screened from the OMIM database and GeneCards database. The intersection targets of QYF and PAD were screened out, and then the enrichment analysis of the signal pathways was conducted. Open field experiments and the O maze experiment were used to verify the pharmacodynamic effect of QYF in the treatment of PAD rats, and the serum neurotransmitter level was detected. The results showed that QYF may be have the function of regulating steroid hormone signaling pathways such as steroid hormone synthesis, ovarian steroidogenesis and estrogen signaling pathways, regulating central neurotransmitters signaling pathways including tryptophan metabolism pathways, Toll-like receptor signaling pathways, TGF-β signaling pathway and regulating inflammatory response pathways including NF-κB signaling pathway, Apelin signaling pathway, MAPK signaling pathway, TNF signaling pathway, and FOXO signaling pathway. All of the signaling pathways were related to PAD, which indicated that QYF may have the role of treating PAD. Qingyan Formula 70% ethanol extract(QYFE) revealed anxiolytic effects by extending the activity time and distance of anxious ovariectomized(OVX) rats in the central field of open field experiments, and increasing the duration and activity distance of anxious OVX rats in the open arm area in O maze experiments. QYFE increased serum serotonin/5-hydroxytryptamine(5-HT), and gamma aminobutyric acid(GABA) levels in anxious OVX rats, significantly reduced glutamate(Glu) and norepinephrine(NA) levels, and reversed the disturbance of excitatory/suppressive transmitter balance caused by OVX, which may be related to its effect on alleviating anxiety disorder in OVX rats.

Transcriptome Profiling Associated with Carcass Quality of Loin Muscles in Crossbred Pigs.

Simple SummaryCarcass quality traits, such as lean depth and loin depth, are of extreme economic importance for the swine industry. This study aimed to identify the gene expression pattern related to carcass quality in crossbred pigs ((Landrace × Yorkshire) × Duroc). In total, 20 crossbred pigs were used in this study and divided into two groups based on the loin muscle quality grade. Total RNA samples extracted from the loin muscles of both groups were submitted for RNA-seq. Differentially expressed gene (DEG) analysis of the two groups revealed 282 up-regulated and 189 down-regulated genes (p ≤ 0.01), linked to tissue development, striated muscle tissue development, tissue morphogenesis, and lipid metabolic process gene ontology (GO) terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis highlighted genes related to the calcium signaling pathway, apelin signaling pathway, and the mammalian target of rapamycin (mTOR) signaling pathway. We constructed an expressed gene catalog, which may serve as a resource for genomic studies focused on uncovering the molecular mechanisms underlying carcass quality in crossbred pigs.Carcass quality traits, such as lean depth and loin depth, are of extreme economic importance for the swine industry. This study aimed to identify the gene expression pattern related to carcass quality in crossbred pigs ((Landrace × Yorkshire) × Duroc). In total, 20 crossbred pigs were used in this study and they were divided into two groups (class I grade, n = 10; class II grade, n = 10) based on the carcass grades. Total RNA samples extracted from the loin muscles of both groups were submitted for RNA-seq. The quality assessment of the sequencing reads resulted in 25,458 unigenes and found 12,795 candidate coding unigenes with homology to other species after annotation. Differentially expressed gene (DEG) analysis of the two groups revealed 282 up-regulated and 189 down-regulated genes (p ≤ 0.01), linked to tissue development, striated muscle tissue development, tissue morphogenesis, and lipid metabolic process gene ontology (GO) terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis highlighted genes related to the calcium signaling pathway, melanogenesis, the sphingolipid signaling pathway, the apelin signaling pathway, and the mTOR signaling pathway. We constructed an expressed gene profile, which may serve as a resource for genomic studies focused on uncovering the molecular mechanisms underlying carcass quality in crossbred pigs.

Pioglitazone protects blood vessels through inhibition of the apelin signaling pathway by promoting KLF4 expression in rat models of T2DM.

Apelin, identified as the endogenous ligand of APJ, exerts various cardiovascular effects. However, the molecular mechanism underlying the regulation of apelin expression in vascular cells is poorly described. Pioglitazone (PIO) and Krüppel-like factor 4 (KLF4) exhibit specific biological functions on vascular physiology and pathophysiology by regulating differentiation- and proliferation-related genes. The present study aimed to investigate the roles of PIO and KLF4 in the transcriptional regulation of apelin in a high-fat diet/streptozotocin rat model of diabetes and in PIO-stimulated vascular smooth muscle cells (VSMCs). Immunohistochemistry, qRT-PCR, and Western blotting assays revealed that the aorta of the Type 2 diabetes mellitus (T2DM) rat models had a high expression of apelin, PIO could decrease the expression of apelin in the PIO-treated rats. In vitro, Western blotting assays and immunofluorescent staining results showed that the basal expression of apelin was decreased but that of KLF4 was increased when VSMCs were stimulated by PIO treatment. Luciferase and chromatin immunoprecipitation assay results suggested that KLF4 bound to the GKLF-binding site of the apelin promoter and negatively regulated the transcription activity of apelin in VSMCs under PIO stimulation. Furthermore, qRT-PCR and Western blotting assay results showed that the overexpression of KLF4 markedly decreased the basal expression of apelin, but the knockdown of KLF4 restored the PIO-induced expression of apelin. In conclusion, PIO inhibited the expression of apelin in T2DM rat models to prevent diabetic macroangiopathy, and negatively regulated the gene transcription of apelin by promoting transcription of KLF4 in the apelin promoter.

Identification of molecular correlations of RBM8A with autophagy in Alzheimer's disease.

Our previous studies revealed RBM8A may play a role in various progressive neurological diseases. The present study aimed to explore the role of RBM8A in Alzheimer's disease (AD). RBM8A is significantly down-regulated in AD. Interestingly, 9186 differentially expressed genes are overlapped from comparisons of AD versus control and RBM8A-low versus RBM8A-high. Weight gene correlation analysis was performed and 9 functional modules were identified. Modules positively correlated with AD and RBM8A-low are significantly involved in the RAP1 signaling pathway, PI3K−AKT signaling pathway, hematopoietic cell lineage, autophagy and APELIN signaling pathway. Fifteen genes (RBM8A, RHBDF2, TNFRSF10B, ACP1, ANKRD39, CA10, CAMK4, CBLN4, LOC284214, NOVA1, PAK1, PPEF1, RGS4, TCEB1 and TMEM118) are identified as hub genes, and the hub gene-based LASSO model can accurately predict the occurrence of AD (AUC = 0.948). Moreover, the RBM8A-module-pathway network was constructed, and low expression of RBM8A down-regulates multiple module genes, including FIP200, Beclin 1, NRBF2, VPS15 and ATG12, which composes key complexes of autophagy. Thus, our study supports that low expression of RBM8A correlates with the decrease of the components of key complexes in autophagy, which could potentially contribute to pathophysiological changes of AD.