Soluble Isoforms Research Articles

IL-23/IL-17 axis and soluble receptors isoforms sIL-23R and sIL-17RA in patients with rheumatoid arthritis-presenting periodontitis.

BackgroundRheumatoid arthritis (RA) and periodontitis (P) are chronic inflammatory diseases characterized by joint and radiographic bone loss, respectively. IL‐23 and IL‐17 have an essential role in the immunopathogenesis of RA, and P. IL‐23 stimulates Th17 cells through which produces IL‐17, IL‐21, and RANKL. IL‐17 stimulates fibroblasts to produce RANKL, which initiates bone loss in the joints in RA and the periodontal tissue in periodontitis. The aim of this study was to determine the expression pattern of IL‐23/IL‐17 axis and soluble receptors isoforms sIL‐23R and sIL‐17RA of patients with RA presenting P (RAP).Material and methodsHealthy subjects (HS) (n = 42), patients with P (n = 40), RA (n = 20), and patients with RAP (n = 40) were included. Plasma samples were obtained to evaluate the IL‐23, IL‐17A, sIL‐23R, and sIL‐17RA by ELISA technique. A nonparametric Mann‐Whitney U test was used to compare the differences between groups. A Chi‐square was used to compare gender, grade and stage of periodontitis, and DAS28‐ESR between the groups. Spearman's rank correlation coefficient was used to study the association between the molecules and clinical parameters.ResultsIL‐23 levels were increased in the RAP group, and lower sIL‐23R levels were found in the RAP groups. However, IL‐17A was lower in the P and RAP group but not in RA patients. RAP group showed a decrease IL‐17A levels in advanced stages of the periodontal disease.ConclusionThese results suggest that IL‐23 and IL‐17A tend to downregulate their expression patterns when patients present both rheumatoid arthritis and periodontitis.

B-PO04-137 SOLUBLE ST-2 LEVELS ARE ELEVATED IN PATIENTS WITH ATRIAL FIBRILLATION

Studies show the association between inflammatory chemokines and atrial fibrillation (AF). Factors, such as CRP, IL-6 and TNF-α, are a few that are well-known to be increased in AF patients. However, there is a paucity of knowledge regarding sST2, a soluble isoform of ST2, known to prevent the NF-κB cell signaling pathway, in AF. To study the relationship between specific chemokines and AF, confirming known inflammatory chemokine associations (CRP, IL-6, MMP2, VCAM-1) and investigating novel ones (sST2). Between July 2018 and June 2019, blood was drawn from consenting subjects 18 to 89 years of age with AF, other atrial arrhythmias, or other cardiovascular disease from a single institution. Patients with congenital heart disease, and end-stage renal disease on hemodialysis, were excluded. Plasma samples were sorted into patients with and without AF. The following chemokines were measured by flow cytometry using the BioLegend LEGENDplex Flow Assay Kit and compared between subjects with and without AF: MCP-1, IL-1B, TNF-α, sST2, IF-γ, VEGF, adiponectin, CRP, MMP2, leptin, I-CAM1, VCAM-1, MMP9. A two-sample t-test was used to compare the groups. Soluble ST2 was significantly higher in AF patients (n=51, 1033 ng/mL, p-value <0.0001) than those without AF (n=65, 862.8 ng/mL). Uniquely, I-CAM1 (AF 868.3 ng/mL, No AF 1146 ng/mL, p-value 0.02), leptin (AF 9227 ng/mL, No AF 14607 ng/mL, p-value 0.01), and IF-γ (AF 20.76, No AF 28.67 ng/mL, p-value <0.0001) were reduced in patients with AF. As expected, IL-6 (AF 17.06 ng/mL, No AF 9.74 ng/mL, p-value <0.0001), CRP (AF 16477 ng/mL, No AF 6547 ng/mL, p-value <0.0001), VCAM-1 (AF 155299 ng/mL, no AF 104246 ng/mL, p-value 0.001), and MMP-2 (AF 224195 ng/mL, No AF 147037 ng/mL, p-value 0.0003) were significantly higher in AF patients. Certain signaling chemokines may predict high risk of developing AF and/or target AF interventions in the future. Interestingly, sST2 is significantly higher in AF patients, though other inflammatory counterparts, such as I-CAM1, leptin and IF-γ, have an inverse association. This suggests that while certain inflammatory ligands may precipitate AF, others may have a protective role.

Leveraging NKG2D Ligands in Immuno-Oncology.

Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.

Enhanced Transcriptomic Resilience following Increased Alternative Splicing and Differential Isoform Production between Air Pollution Conurbations

Adverse health outcomes caused by ambient particulate matter (PM) pollution occur in a progressive process, with neutrophils eliciting inflammation or pathogenesis. We investigated the toxico-transcriptomic mechanisms of PM in real-life settings by comparing healthy residents living in Beijing and Chengde, the opposing ends of a well-recognised air pollution (AP) corridor in China. Beijing recruits (BRs) uniquely expressed ~12,000 alternative splicing (AS)-derived transcripts, largely elevating the proportion of transcripts significantly correlated with PM concentration. BRs expressed PM-associated isoforms (PMAIs) of PFKFB3 and LDHA, encoding enzymes responsible for stimulating and maintaining glycolysis. PMAIs of PFKFB3 featured different COOH-terminals, targeting PFKFB3 to different sub-cellular functional compartments and stimulating glycolysis. PMAIs of LDHA have longer 3′UTRs relative to those expressed in Chengde recruits (CRs), allowing glycolysis maintenance by enhancing LDHA mRNA stability and translational efficiency. PMAIs were directly regulated by different HIF-1A and HIF-1B isoforms. BRs expressed more non-functional Fas isoforms, and a resultant reduction of intact Fas proportion is expected to inhibit the transmission of apoptotic signals and prolong neutrophil lifespan. BRs expressed both membrane-bound and soluble IL-6R isoforms instead of only one in CRs. The presence of both IL-6R isoforms suggested a higher migration capacity of neutrophils in BRs. PMAIs of HIF-1A and PFKFB3 were downregulated in Chronic Obstructive Pulmonary Disease patients compared with BRs, implying HIF-1 mediated defective glycolysis may mediate neutrophil dysfunction. PMAIs could explain large variances of different phenotypes, highlighting their potential application as biomarkers and therapeutic targets in PM-induced diseases, which remain poorly elucidated.

Glutathione-S-transferases genes-promising predictors of hepatic dysfunction.

One of the most commonly known genes involved in chronic diffuse liver diseases pathogenesis are genes that encodes the synthesis of glutathione-S-transferase (GST), known as the second phase enzyme detoxification system that protects against endogenous oxidative stress and exogenous toxins, through catalisation of glutathione sulfuric groups conjugation and decontamination of lipid and deoxyribonucleic acid oxidation products. The group of GST enzymes consists of cytosolic, mitochondrial and microsomal fractions. Recently, eight classes of soluble cytoplasmic isoforms of GST enzymes are widely known: α-, ζ-, θ-, κ-, μ-, π-, σ-, and ω-. The GSTs gene family in the Human Gene Nomenclature Committee, online database recorded over 20 functional genes. The level of GSTs expression is considered to be a crucial factor in determining the sensitivity of cells to a broad spectrum of toxins. Nevertheless, human GSTs genes have multiple and frequent polymorphisms that include the complete absence of the GSTM1 or the GSTT1 gene. Current review supports the position that genetic polymorphism of GST genes is involved in the pathogenesis of various liver diseases, particularly non-alcoholic fatty liver disease, hepatitis and liver cirrhosis of different etiology and hepatocellular carcinoma. Certain GST allelic variants were proven to be associated with susceptibility to hepatological pathology, and correlations with the natural course of the diseases were subsequently postulated.

Alternatively Spliced Tissue Factor Levels in the Plasma of Patients with Liver Fibrosis, Liver Cirrhosis, and Hepatocellular Carcinoma: A Single-Center Retrospective Study

Background:Chronic liver diseases (CLD) such as chronic viral hepatitis, metabolic liver diseases, alcoholic liver disease, primary and secondary hemochromatosis, Wilson's disease, and cholestatic or autoimmune liver diseases are the major cause of morbidity and mortality worldwide ranking as the 12th leading cause of overall mortality. CLD induce liver fibrosis characterized by the accumulation of extracellular matrix proteins, such as collagen, in the liver interstitial space. If untreated, liver fibrosis may evolve to end stage cirrhosis, which is associated with a high risk of developing hepatocellular carcinoma (HCC) and liver failure.CLD is accompanied by changes in the coagulation system that embrace not only hypo-, but also hyper-coagulability. In this rebalanced situation, hypercoagulability can contribute, like genetic predisposition or environmental factors, to the progression of fibrosis and complications of cirrhosis.Full-length tissue factor (TF), a glycosylated transmembrane protein, is the only initiator of the extrinsic coagulation cascade in vivo. It is elevated in patients with pancreatic cancer, blood disorders, diabetes, and cardiovascular diseases. During TF pre-mRNA processing, exon 5 can be spliced out, resulting in a frameshift that yields to a soluble TF isoform called alternatively spliced TF (asTF). While its involvement in thrombosis and/or hemostasis is still debated, asTF induces angiogenesis and was found highly expressed in pancreatic ductal adenocarcinoma and promoted cancer progression. More recently, it was reported that asTF mRNA expression levels may constitute a prognostic marker in human gastric cancer.MethodsIn a single-center study, we analyzed retrospectively asTF plasma levels in healthy subjects, patients with liver fibrosis, liver cirrhosis, and HCC. asTF plasma levels were measured using a sandwich enzyme-linked immunosorbent assay (ELISA). In addition, immunostaining for asTF was performed in HCC tissue biopsies using an anti-asTF specific antibody. Values were expressed as mean±SD.ResultsThe patients with HCC displayed the highest mean asTF levels (n=128, 552.2±469.7pg/ml), followed by those with liver fibrosis (n=70, 524.4±453.9pg/ml) and liver cirrhosis (n=62, 357.2±389.8pg/ml). There was a significant difference in asTF levels between healthy subjects (n=60, 127.3±136.3pg/ml) and patients with liver fibrosis (p<0.0001), healthy subjects and cirrhotic patients (p<0.0001), healthy subjects and HCC patients (p<0.0001), cirrhotic patients and HCC patients (p=0.004) and between fibrotic and cirrhotic patients (p=0.03). Between fibrotic patients and HCC patients, no significant difference in asTF levels was detectable (p=0.70). There were no effects of gender, age and underlying liver disease (hepatitis B and C, alcohol abuse, nonalcoholic steatohepatitis) in each patient group on asTF levels. Furthermore, asTF levels did not correlate with Child-Pugh score, grade of liver fibrosis, and HCC tumor size. Preliminary results indicate that asTF is expressed in liver tissue by hepatocytes, cholangiocytes, and endothelial cells.ConclusionOur study revealed that asTF plasma levels were elevated not only in patients with HCC, but also in patients with cirrhosis and even in those with liver fibrosis. This suggests that asTF is produced during fibrotic remodeling of the liver. The lack of correlation between grade of liver fibrosis and asTF levels could be due to the fact that asTF levels did not correlate with the amount of fibrosis deposited in the parenchyma but with the process of producing the fibrosis. In summary, our observations suggest that asTF might play a role in hepatic fibrogenesis. DisclosuresNo relevant conflicts of interest to declare.

Evidence for a shift in placental HLA-G allelic dominance and the HLA-G isoform profile during a healthy pregnancy and preeclampsia†.

Human leukocyte antigen (HLA)-G, which belongs to a nonclassical class Ib major histocompatibility complex gene family expressed by placental trophoblast cells, plays a central role in establishing tolerance to the semiallogeneic fetus and in placentation. HLA-G exists in different soluble or membrane-bound isoforms. Preeclampsia, a major cause of fetal and maternal morbidity and mortality, has been linked to insufficient placentation and an altered immune response in pregnancy, including altered HLA-G expression. The 14bp insertion/deletion polymorphism in the 3' untranslated region of the gene and the isoform profile may affect HLA-G expression. The aim of the current pilot study was to characterize the expression patterns of HLAG mRNA, protein, and isoform profile in uncomplicated term pregnancies and in cases of preeclampsia. Maternal sHLA-G mRNA and protein levels were slightly reduced in preeclampsia. No difference was found for placental blood, and no correlation between peripheral and placental sHLA-G levels was found. We observed no association between neither fetal nor maternal HLA-G 14bp insertion/deletion genotypes and preeclampsia, nor a significant difference in isoform profiles. However, in HLA-G 14bp insertion/deletion heterozygous placental samples, we observed abundant HLA-G1 14bp insertion allele expression in the term placentae, which is contrary to previous findings in first trimester trophoblast. Increased HLA-G1 14bp insertion allele expression in the placenta was associated with reduced levels of placental sHLA-G and an altered isoform profile with increased relative levels of HLA-G1 and -G5 and reduced levels of HLA-G3. The results indicate that an allelic shift in heterozygous individuals could represent a novel regulatory pathway.

MO630SNOGO-B IS AN IMPORTANT CONTRIBUTOR TO GLOMERULAR ENDOTHELIAL CELL STABILITY AND VASCULAR REMODELLING INTERVENING ON VEGFA/VEGFR2 SIGNALLING

Abstract Background and Aims Nogo-B is an endoplasmic reticulum protein present as a full length and circulating soluble isoform (sNogo-B) corresponding to the first ∼200aa of the N-terminus. Nogo-B is expressed in glomerular endothelial cells (GECs) and is downregulated in the diabetic glomeruli; its repletion, ameliorates diabetic glomerulopathy. However, the precise biological role of Nogo-B and its soluble form in GEC is not well understood. We hypothesise that sNogo-B could modulate VEGFA/VEGFR2 signalling, and vascular remodelling resulting in improved GECs health and vascular remodelling (vessel repair/new vessel formation). We predict that this effect may be mediated by changes in VEGFA/VEGFR2 signalling; a critical signalling pathway which regulates blood vessel function in physiology and disease. Method For this experiment we used human conditionally immortalised GECs. Cells were used after differentiation at 37°. GECs were infected with adenovirus vector expressing sNogo-B or identical vector lacking sNogo-B cDNA (control vector). Cells were serum starved (4 hours, FBS 2%) and exposed to VEGFA (50 ng/ml) for 5’ 10’ 15’ min and VEGFR2 phosphorylation assessed with western immunoblotting. Results VEGFA-Mediated VEGFR2 phosphorylation was upregulated in wild-type GECs after 15 min VEGFA incubation (P&amp;lt;0.05) n=4. sNogo-B overexpression upregulated the sNogo-B protein level in the supernatant by 10 fold and prevented VEGFA/VEGFR2 phosphorylation in human immortalized glomerular endothelial cells(P&amp;lt;0.05). Conclusion sNogo-B prevents the VEGFA mediated VEGFR2 phosphorylation in GECs. Upregulation of VEGFA/VEGFR2 signalling has been implicated in diabetic glomerulopathy. sNogo-B inhibition of VEGFA/VEGFR2 signalling opens new investigations looking at the potential role of sNogo-B as therapeutic target in diabetic nephropathy.

S-VCAM-1 is an independent predictor of all-cause mortality in patients with dilated cardiomyopathy and hypokinetic non-dilated cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): University Medicine Munich University Medicine Greifswald Introduction The vascular cell adhesion molecule-1 (VCAM-1) is overexpressed in a number of different inflammatory processes on activated endothelium. This could be shown in both a mouse model for autoimmune myocarditis and in human heart tissue from patients with lymphocytic myocarditis. In addition to the tissue-bound one, a soluble isoform of VCAM-1 (s-VCAM-1) can also be detected in the blood. Higher levels have been associated with worse clinical outcome in chronic heart failure patients of different etiology and other patient groups. Purpose Since both inflammation and fibrosis are key processes involved in the pathogenesis of dilated cardiomyopathy (DCM) and hypokinetic non-dilated cardiomyopathy (HNDC), we aimed to investigate the prognostic value of s-VCAM-1 plasma levels for survival in a large cohort of DCM and HNDC patients. Methods The cohort comprised of patients with a primary diagnosis of DCM, defined as reduced left ventricular ejection fraction (LVEF &amp;lt;45%), increased left ventricular enddiastolic diameter according to HENRY score (LVEDD &amp;gt;117%) at time of diagnosis as well as HNDC, defined as a reduced left ventricular ejection fraction (LVEF &amp;lt;45%) but no increased LVEDD according to HENRY score (LVEDD &amp;lt; =117%). Exclusion criteria were primary valvular diseases (≥ second degree), acute myocarditis, cancer, chronic alcoholism, coronary artery disease with epicardial stenosis &amp;gt;50%, peripheral artery occlusive disease, known auto-immune disease and heart failure of other origins. Levels of s-VCAM-1 were measured in human plasma using an enzyme-linked immunosorbent assay (R&amp;D Systems, USA). A Cox proportional hazard model for the association between s-VCAM-1 and all-cause mortality was adjusted for age, sex, time since symptom-onset, LVEF, kidney function (eGFR-CKDEPI), CRP and NT-proBNP. Results A total of 334 DCM patients were included in this single-center cohort (78.4 % males) with a mean age of 54.0 years [interquartile range [IQR] 47.0, 63.2). On average time since symptom onset was 1.5 years (IQR 0.1, 1.1), LVEF 30.7 % (IQR 25, 37), LVEDD 67.1 mm (IQR 62, 72). During a median follow-up of 12.4 years (IQR 10.1, 13.9), a total of 118 (35.3 %) patients died. Multivariable-adjusted cox regression model revealed a significantly increased all-cause mortality risk with increasing levels of s-VCAM-1 (p for trend =0.039), (hazard ratio [HR] 1.00045 (Conf. Interval 1.00002, 1.00087) for VCAM increase of 1 ng/mL, for increase of 100 ng/ml HR 1.046 (Conf- interval 1.002, 1.091), for increase of 1000ng/ml HR 1.57 (Conf_interval 1.02-2.41) (Kaplan Meier survival estimates see Figure 1, median s-VCAM-1 = 664 ng/ml, IQR 515,874). Conclusions s-VCAM-1 predicts long-term survival in DCM patients independent of NT-pro-BNP and other risk determinants. Further research needs to evaluate whether this biomarker proves useful in monitoring and planning management of DCM and HNDC patients (e.g. more intensive management in high-risk patients). Abstract Figure. Kaplan-Meier survival estimates

ST2L expression on monocyte subsets is associated with the severity of acute coronary syndrome and plaque vulnerability

IntroductionPro-inflammatory monocyte infiltration and conversion to macrophages is an established risk factor for acute coronary syndrome (ACS) and plaque instability visualized by optical coherence tomography (OCT). Increased serum levels of soluble ST2 isoform (sST2) predict poor outcomes in patients with ACS. However, whether the transmembrane ST2 isoform (ST2L) could early assess the severity of ACS and the vulnerability of plaques remains elusive.Material and methodsFlow cytometry and ELISA were performed to evaluate the levels of ST2L on different monocyte subsets in ACS patients and serum concentration of sST2. Bone marrow-derived macrophages (BMDMs) were isolated and polarized to M1 or M2 phenotype, which levels of ST2L expression were detected by Quantitative RT-PCR and Immunoblots.ResultsThe proportion and absolute number of ST2L on monocyte subsets 1 (Mon1) and Mon2 significantly declined, accompanied with increased serum sST2, in ACS patients comparing with controls. However, only proportion of ST2L+/Mon2 remained closely associated with the severity of atherosclerotic plaques. Patients with thin cap fibrous atheroma (TCFA) calculated by OCT had lower absolute number (Non-TCFA 1.26×104 ± 0.63×104/ml vs. TCFA 0.92×104 ± 0.37×104/ml, P = 0.034) and lower percentage (Non-TCFA 17.65% ± 3.10% vs. TCFA 13.17% ± 2.29%, P &lt; 0.001) of ST2L+/Mon2 than those without TCFA. In multivariate analysis, the proportion of ST2L+/Mon2 was considered as an independent determinant for TCFA. In BMDMs, sST2 and ST2L were highly expressed in M2 macrophage.ConclusionsCompared to circulating sST2, the proportion of ST2L expressing on Mon2 subsets was more strongly associated with the severity of ACS and plaque vulnerability.

In Search of a Role for Extracellular Purine Enzymes in Bone Function.

Bone is one of the major tissues that undergoes continuous remodeling throughout life, thus ensuring both organic body growth during development and protection of internal organs as well as repair of trauma during adulthood. Many endogenous substances contribute to bone homeostasis, including purines. Their role has increasingly emerged in recent decades as compounds which, by interacting with specific receptors, can help determine adequate responses of bone cells to physiological or pathological stimuli. Equally, it is recognized that the activity of purines is closely dependent on their interconversion or metabolic degradation ensured by a series of enzymes present at extracellular level as predominantly bound to the cell membrane or, also, as soluble isoforms. While the effects of purines mediated by their receptor interactions have sufficiently, even though not entirely, been characterized in many tissues including bone, those promoted by the extracellular enzymes providing for purine metabolism have not been. In this review, we will try to circumstantiate the presence and the role of these enzymes in bone to define their close relationship with purine activities in maintaining bone homeostasis in normal or pathological conditions.

The Duplicitous Nature of ACE2 in COVID-19 Disease.

The Duplicitous Nature of ACE2 in COVID-19 Disease.

Imbalance of growth factors mRNA expression associated with oxidative stress in the early pregnancy loss

Objective The aim of this study was to examine the role of growth factors associated with angiogenesis and oxidative stress in the pathogenesis of spontaneous miscarriage. Methods We performed a comparative mRNA expression analysis of VEGF, PlGF, Flt-1, Angiogenin and Endoglin using Real-Time PCR, in the placenta and decidua collected from 12 patients presenting with spontaneous abortion and from 14 women undergoing induced abortion, during the first and second trimester of pregnancy. Results The mRNA expression of Flt-1 was significantly upregulated in the placenta of spontaneous abortions (5.17-fold, IQR: 2.72–9.11, p < 0.01). The placental expression of the soluble isoforms of Flt-1, sFlt-1 e15a and sFlt-1 i13, was also significantly upregulated in spontaneous abortions (sFlt-1 e15a: 2.35-fold, IQR: 0.98–2.83, p < 0.01; sFlt-1 i13: 3.47-fold, IQR: 2.37–5.08, p < 0,05). Placental tmFlt-1, PlGF and Endoglin showed a tendency of higher expression levels in spontaneous abortions, although they did not reach statistical significance (tmFlt-1: 7.42-fold, IQR: 3.58–14.32; PlGF: 2.36-fold, IQR: 0.90–4.12; Endoglin: 1.97-fold, IQR: 1.18–2.43). VEGF and Angiogenin mRNA expression in induced, as well as in spontaneous abortions, did not convey any statistically significant difference. In the decidua, the expression levels of Flt-1 and its splice variants sFlt-1 e15a, sFlt-1 i13 and tmFlt-1 did not show any statistically significant differences, as was the case for the rest of the herein examined growth factors. Conclusions In this study, we observed higher levels of sFlt-1 mRNA expression in the placenta of spontaneous abortions, while expression of other growth factors in placenta and decidua remained constant. This suggests that an imbalance of sFlt-1 expression in the placenta might contribute to the pathogenesis of spontaneous abortion, probably via oxidative stress, providing a possible biomarker for prompt identification of this condition.

URINARY SOMATIC ANGIOTENSIN CONVERTING ENZYME AND SOLUBLE N-DOMAIN ISOFORMS (90 AND 65KDA) EXPRESSION AND CORRELATION WITH DIFFERENT NUTRITIONAL STATUS/CARDIOVASCULAR RISK PROFILE

Abstract Objective: Angiotensin converting enzyme (ACE) has been involved in physiopathology of several diseases including hypertension, diabetes, obesity and metabolic syndrome. The somatic ACE weights from 130–190 KDa and presents N- and C-domains. Two soluble N-domain isoforms have been described in human urine, weighting 65 and 90KDa. Studies have supported that N-domain ACE with 90KDa is a biomarker for hypertension, pre-eclampsia and inflammation. We evaluated the expression of somatic ACE and soluble N-domain isoforms (90 and 65KDa) in urine of children and adolescents with different nutritional status and cardiovascular risk profile. Design and method: The participants aged from 6 to 19 years were classified into four groups according to their BMI percentile; underweight (n = 51), normal weight (n = 53), overweight (n = 53) and obese (n = 49). Waist-height-ratio (WHtR) was used to assess cardiovascular risk profile dividing the participants into normal risk (n = 105) and high risk (n = 101). The urines were concentrated 10-fold and dialyzed with Tris-HCl pH 8 and pure water. Then, we performed western blot analysis using 50 mg of lyophilized urinary protein. ACE expression was proved against the polyclonal antibody Y1. Protein detection was performed by chemiluminescent and analysis was done in Image Labâ (BioRad) software utilizing total protein stain for normalization. Results: ACE expression is augmented in obese children when compared with normal weight children (0.09 vs 0.53 arbitrary units, p = 0,04). The higher cardiovascular risk group also presented increased expression of ACE (0.27 vs 0.09 arbitrary units, p = 0.046). The 90KDa N-domain isoform is frequently found in the high cardiovascular risk children (p = 0.02). Additionally, according to Spearman correlation test, the expression of 90KDa N-domain isoform correlates positively with waist circumference (cm), WHtR, BMI percentile and Z-score of BMI. Conclusions: Increased ACE expression in obese children contributes to higher cardiovascular risk once this enzyme biosynthesizes Angiotensin II which promotes blood pressure increase, sympathetic nervous system activation and release of glucocorticoids from adrenal gland. ACE expression is also augmented in children with high cardiovascular risk. Presence of 90 KDa N-domain ACE in urine of children and adolescents is a biomarker of poor prognostic for cardiovascular disease in childhood obesity.

UMBILICAL CORD-DERIVED MESENCHYMAL STROMAL CELLS AS AN ALTERNATIVE TO BONE MARROW IN IMMUNOSUPPRESSIVE THERAPY

<h3>Background</h3> Due to their immunomodulatory characteristics, mesenchymal stromal cells (MSC) have become an option in immunosuppressive therapy. HLA-G expression appears to play a key role in immunomodulation, inhibiting proliferation of immune cells and inducing activation of regulatory cells, modulating host‘s immune response. Thus, identifying a source of MSC with high HLA-G expression can be a good strategy for future treatments. <h3>Methods</h3> MSC from umbilical cord tissue (UCT) (n=3) were compared to a sample of bone marrow (BM) (n=1) under the following growing conditions: (i) standard cultivation; (ii) stimulation of interferon-γ (IFN-γ); and (iii) hypoxia condition (2% O2). Expression of CD14, CD73, CD34, CD19, CD166, CD29, CD45, CD90, HLA-DR and CD105 markers and co-stimulatory CD40, CD80 and CD86 markers were analyzed. The analysis of CD152 molecule (intracellular isoform) and membrane HLA-G1 isoform was performed by flow cytometry to study the expression of immunosuppressive molecules. The analysis of soluble HLA-G5 isoform was performed by enzyme-linked immunosorbent assay (ELISA). For analysis of the immunosuppressive potential of MSCs, a lymphocyte (PBMCs) inhibition assay was performed in proportions 1:2 and 1:10 (MSC:PBMC). <h3>Results</h3> The expression of cellular markers was following that required by the International Society for Cell and Gene Therapy. All samples showed negative expression for co-stimulatory markers CD40, CD80 and CD86 and positive expression for CD152. CD152 and HLA-G1 molecules were more expressed by UC when grown under normal conditions (<i>p</i>=0.0018 and <i>p</i>=0.0003, respectively) or hypoxia (<i>p</i>=0.0057 and <i>p</i><0.0001, respectively), but were similar when grown with IFN-γ (<i>p</i>=0.2888 and <i>p</i>=0.2307, respectively). The expression of soluble isoform HLA-G5 was similar by UC and BM. UC and BM showed similar inhibition of PBMCs in standard culture or hypoxia. However, when stimulated with IFN-γ, BM showed greater inhibitory capacity in the proportion 1:2 (<i>p</i>=0.0011) and 1:10 (<i>p</i>=0.0177). IFN-γ increases inhibition of PBMC by BM in relation to standard culture (<i>p</i>=0.0219). <h3>Conclusion</h3> We observed that IFN-γ was able to trigger an increase in inhibitory capacity of BM MSC, but not in UCT-MSC, being indicated in cultivation of BM MSC used for immunosuppressive therapy. In addition, UCT-MSC proved to be an efficient alternative to BM MSC. UCT-MSC showed an advantage in the expression of immunosuppressive

The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, Canada

BackgroundAngiotensin converting enzyme 2 (ACE2) protein serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 transcription in people tested for COVID-19 and the relationship between ACE2 transcription and SARS-CoV-2 viral load, while adjusting for expression of: (i) the complementary protease, Transmembrane serine protease 2 (TMPRSS2), (ii) soluble ACE2, (iii) age, and (iv) biological sex. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts. MethodsA cross-sectional study of n = 424 “participants” aged 1–104 years referred for COVID-19 testing was performed in British Columbia, Canada. Patients who tested positive for COVID-19 were matched by age and biological sex to patients who tested negative. Viral load and host gene expression were assessed by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection. FindingsAnalysis showed no association between age and nasopharyngeal ACE2 transcription in those who tested negative for COVID-19 (P = 0•092). Mean relative transcription of transmembrane (P = 0•00012) and soluble (P<0•0001) ACE2 isoforms, as well as TMPRSS2 (P<0•0001) was higher in COVID-19-negative participants than COVID--19 positive ones, yielding a negative correlation between targeted host gene expression and positive COVID-19 diagnosis. In bivariate analysis of COVID-19-positive participants, transcription of transmembrane ACE2 positively correlated with SARS-CoV-2 viral RNA load (B = 0•49, R2=0•14, P<0•0001), transcription of soluble ACE2 negatively correlated (B= -0•85, R2= 0•26, P<0•0001), and no correlation was found with TMPRSS2 transcription (B= -0•042, R2=<0•10, P = 0•69). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 transcription (Β= 0•89, 95%CI: [0•59 to 1•18]), while transcription of the soluble isoform appears to protect against high viral RNA load in the upper respiratory tract (Β= -0•099, 95%CI: [-0•18 to -0•022]). InterpretationNasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2. FundingThis project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.

VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6–12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.

Endoglin Expression and Surface Renewal in Mesenchymal Stem Cells and Endothelial Cells

Endoglin (CD105) is one of the major marker antigens of mesenchymal stem cells (MSC) and endothelial cells. While the functions of endoglin in endothelial cells have been widely declared, little is known about its role in MSC biology. Here, we present a comparative study of CD105 expression, interna-lization and shedding by human endothelial cell line EA.hy926 and human adipose tissue-derived MSC from various sources. While the fraction of CD105-positive cells in all MSC cultures and EA.hy926 endothelial cells was consistently high, over 97% of the population, the density of CD105 molecules on the surface of MSC isolated from visceral and subcutaneous adipose tissue was substantially different. The total expression level of endoglin mRNA in MSC and endothelial cells was similar, whereas the contribution of the transcripts that yield a short CD105 isoform was higher in endothelial cells. Using a set of monoclonal antibodies (mAbs) directed against different endoglin epitopes, we revealed significant differences in the dynamics of CD105 metabolism on the membranes of endothelial cells and MSC. On EA.hy926 endothelial cells, CD105 molecules bound with antibodies were internalized and remained in the perinuclear space. In MSC cultures, on the contrary, the CD105-mAbs complexes were not subjected to endocytosis and remained on the cell membrane for a long time. We demonstrated that MSC, similarly to the endothelial cells, do shed the extracellular endoglin fragment into the environment as a soluble CD105 isoform. The shedding process in MSC was, however, substantially less intensive compared with the endothelial cells. Thus, we revealed, for the first time, that in MSC, unlike the endothelial cells, endoglin persists on cell surface for a long time and is not interna-lized after binding with antibodies. Endoglin shedding from MSC surface and the concomitant generation of the soluble endoglin form were also demonstrated for the first time.

Abstract P657: Soluble Receptor for Advanced Glycation End Products and Subtypes Are Protective Biomarkers of Functional Outcome but Not Those of Recurrence in Acute Ischemic Stroke

Background and purpose: Soluble isoforms of receptor for advanced glycation end products (sRAGE) and subtypes have been recognized as contradictory biomarkers of ischemic stroke. We sought to investigate whether the plasma levels of sRAGE and subtypes can predict unfavorable outcome and recurrence in acute ischemic stroke patients. Methods: The data used in this study was from the Third China National Stroke Registy (CNSR III), which was a nationwide, prospective cohort study to register acute ischemic stroke and transient ischemic attack patients. Plasma levels of sRAGE and subtypes were tested by enzyme-linked immunoabsorbent assay (ELISA) method and demonstrated by quartiles. Cox proportional hazards model was used to analysis the association of sRAGE and subtypes with unfavorable outcomes or stroke recurrence at 3- and 12-month in acute ischemic stroke patients, respectively. Unfavorable outcome was defined as modified Rankin Scale (mRS) 3-6. Results: Three thousand one hundred and eighty-nine acute ischemic stroke patients were included and tested for plasma level of sRAGE and subtypes (cRAGE and esRAGE) in this study. Mean age was 62.8±11.5 years and 2161 (67.8%) were male. At 3- and 12-month, there were 426 (13.5%) and 409 (13.1%) patients with an unfavorable outcome and 185 (5.9%) and 293 (9.2%) patients with stroke recurrence, respectively. Refered by quartile one of sAGRE, the adjusted hazard ratios (aHRs) and 95% confidence intervals (95% CIs) of unfavorable outcomes in quartile two to four were 0.81 (0.59-1.10), 0.66 (0.48-0.92) and 0.65 (0.47-0.91) at 3-month and 0.79 (0.58-1.08), 0.61 (9.44-0.85) and 0.66 (0.48-0.91) at 12-month; those of stroke recurrence in quartile two to four were 0.99 (0.66-1.49), 0.99 (0.66-1.49) and 1.02 (0.68-1.54) at 3-month and 1.05 (0.76-1.45), 0.95 (0.68-1.32) and 1.07 (0.77-1.48) at 12-month, respectively. Same data trends were found in subtypes cRAGE and esRAGE. Conclusion: Plasma levels of sRAGE and subtypes were protective biomarkers of unfavorable outcomes but not those of stroke recurrence in acute ischemic stroke patients.

The miRNA199a/SIRT1/P300/Yy1/sST2 signaling axis regulates adverse cardiac remodeling following MI

Left ventricular remodeling following myocardial infarction (MI) is related to adverse outcome. It has been shown that an up-regulation of plasma soluble ST2 (sST2) levels are associated with lower pre-discharge left ventricular (LV) ejection fraction, adverse cardiovascular outcomes and mortality outcome after MI. The mechanisms involved in its modulation are unknown and there is not specific treatment capable of lowering plasma sST2 levels in acute-stage HF. We recently identified Yin-yang 1 (Yy1) as a transcription factor related to circulating soluble ST2 isoform (sST2) expression in infarcted myocardium. However, the underlying mechanisms involved in this process have not been thoroughly elucidated. This study aimed to evaluate the pathophysiological implication of miR-199a-5p in cardiac remodeling and the expression of the soluble ST2 isoform. Myocardial infarction (MI) was induced by permanent ligation of the left anterior coronary artery in C57BL6/J mice that randomly received antimiR199a therapy, antimiR-Ctrl or saline. A model of biomechanical stretching was also used to characterize the underlying mechanisms involved in the activation of Yy1/sST2 axis. Our results show that the significant upregulation of miR-199a-5p after myocardial infarction increases pathological cardiac hypertrophy by upregulating circulating soluble sST2 levels. AntimiR199a therapy up-regulates Sirt1 and inactivates the co-activator P300 protein, thus leading to Yy1 inhibition which decreases both expression and release of circulating sST2 by cardiomyocytes after myocardial infarction. Pharmacological inhibition of miR-199a rescues cardiac hypertrophy and heart failure in mice, offering a potential therapeutic approach for cardiac failure.