Endothelial Gene Expression Research Articles

Insights into the In Vitro Formation of Apatite from Mg‐Stabilized Amorphous Calcium Carbonate

AbstractA protein‐free formation of bone‐like apatite from amorphous precursors through ball‐milling is reported. Mg2+ ions are crucial to achieve full amorphization of CaCO3. Mg2+ incorporation generates defects which strongly retard a recrystallization of ball‐milled Mg‐doped amorphous calcium carbonate (BM‐aMCC), which promotes the growth of osteoblastic and endothelial cells in simulated body fluid and has no effect on endothelial cell gene expression. Ex situ snapshots of the processes revealed the reaction mechanisms. For low Mg contents (<30%) a two phase system consisting of Mg‐doped amorphous calcium carbonate (ACC) and calcite “impurities” was formed. For high (>40%) Mg2+ contents, BM‐aMCC follows a different crystallization path via magnesian calcite and monohydrocalcite to aragonite. While pure ACC crystallizes rapidly to calcite in aqueous media, Mg‐doped ACC forms in the presence of phosphate ions bone‐like hydroxycarbonate apatite (dahllite), a carbonate apatite with carbonate substitution in both type A (OH−) and type B (PO43−) sites, which grows on calcite “impurities” via heterogeneous nucleation. This process produces an endotoxin‐free material and makes BM‐aMCC an excellent “ion storage buffer” that promotes cell growth by stimulating cell viability and metabolism with promising applications in the treatment of bone defects and bone degenerative diseases.

Covalent grafting of PEG and heparin improves biological performance of electrospun vascular grafts for carotid artery replacement

Rapid endothelialization of small-diameter vascular grafts remains a significant challenge in clinical practice. In addition, compliance mismatch causes intimal hyperplasia and finally leads to graft failure. To achieve compliance match and rapid endothelialization, we synthesized low-initial-modulus poly(ester-urethane)urea (PEUU) elastomer and prepared it into electrospun tubular grafts and then functionalized the grafts with poly(ethylene glycol) (PEG) and heparin via covalent grafting. The PEG- and heparin-functionalized PEUU (PEUU@PEG-Hep) graft had comparable mechanical properties with the native blood vessel. In vitro data demonstrated that the grafts are of good cytocompatibility and blood compatibility. Covalent grafting of PEG and heparin significantly promoted the adhesion, spreading, and proliferation of human umbilical vein endothelial cells (HUVECs) and upregulated the expression of vascular endothelial cell-related genes, as well as increased the capability of grafts in preventing platelet deposition. In vivo assessments indicated good biocompatibility of the PEUU@PEG-Hep graft as it did not induce severe immune responses. Replacement of resected carotid artery with the PEUU@PEG-Hep graft in a rabbit model showed that the graft was capable of rapid endothelialization, initiated vascular remodeling, and maintained patency. This study demonstrates the PEUU@PEG-Hep vascular graft with compliance match and efficacious antithrombosis might find opportunities for bioactive blood vessel substitutes.

Exploring the transcriptomic network of multi-ligand scavenger receptor Stabilin-1- and Stabilin-2-deficient liver sinusoidal endothelial cells.

The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-β/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood.

Fibroblast cell derived extracellular matrix containing electrospun scaffold as a hybrid biomaterial to promote in vitro endothelial cell expansion and functionalization

Biomaterials to be used for vascular tissue engineering must allow attachment, proliferation, and functionalization of vasoactive cells especially endothelial cells. In this study, decellularized L929 fibroblast cell-derived ECM containing electrospun scaffolds were fabricated and their biological response was investigated using rat glomerulus endothelial cells (rGECs). The L929 cells were grown for one week to get cell sheets on PCL membranes followed by decellularization of whole cell sheet-PCL membrane (PCL-ECM) using sodium dodecyl sulfate (SDS)/triton X-100 (TX) or freeze/thaw (F/T)/Deoxyribonuclease cycle to yield the corresponding mechanically stable scaffold. The nucleic acids and structural proteins quantification were performed on various membranes before and after decellularization process. Seeded rGECs on PCL, PCL-ECM (SDS/TX) and PCL-ECM (F/T) membranes were investigated through immunofluorescence and cell proliferation assay. The bio-macromolecules contents on decellularized scaffolds showed diverse outcome because of different decellularization methods used. The hydrophilic PCL-ECM (F/T) scaffold showed the best result by ensuring stability, good cytocompatibility, and interconnections among endothelial cells as was further confirmed by endothelial gene expression analysis. In short, the outcomes of this study may pave the way for the construction of new cell-derived ECM based vascular tissue engineering scaffolds as well as for the development of in vitro models to study endothelial cell function.

Effects of quercetin and metabolites on uric acid biosynthesis and consequences for gene expression in the endothelium

BackgroundUric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. MethodsInhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 μmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. ResultsIn human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3′-sulfate and the gut microbial metabolite 3′,4′-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. ConclusionsWe propose that the main mechanism by which quercetin, as quercetin-3′-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.

Isolation of Highly Purified and Viable Retinal Endothelial Cells

The neonatal mouse retinal vascularization model has been widely used in the vascular biology field to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Recent advances in next-generation sequencing can further elucidate mechanisms of blood vessel formation and remodeling in this, as well as other, vascular development models. However, an optimized method for isolating retinal endothelial cells that limits tissue digestion-induced cell damage is required for next-generation sequencing applications. In this study, we established a method for isolating neonatal retinal endothelial cells that optimizes cell viability and purity. The CD31+/CD45− endothelial cell population was fluorescence-activated cell sorting (FACS)-isolated from digested postnatal retinas, found to be highly enriched for endothelial cell gene expression, and exhibited no change in viability for 60 min post-FACS. Thus, this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. Combining this isolation method with next-generation sequencing will enable further delineation of mechanisms underlying vascular development and maturation.

Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression

Aims/hypothesisReduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs).MethodsTerm placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 Mr dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured.ResultsAll three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 Mr dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold).Conclusions/interpretationDownregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring.Graphical abstract

Quantitative Evaluation of Human Umbilical Vein and Induced Pluripotent Stem Cell-Derived Endothelial Cells as an Alternative Cell Source to Skin-Specific Endothelial Cells in Engineered Skin Grafts.

Objective: We compared the capability of human umbilical vein endothelial cells (HUVECs), induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs), and human dermal blood endothelial cells (HDBECs) to effectively vascularize engineered human skin constructs (HSCs) in vitro and on immunodeficient mice. Approach: We quantified the angiogenesis within HSCs both in vitro and in vivo through computational analyses of immunofluorescent (IF) staining. We assayed with real-time quantitative PCR (RT-qPCR) the expression of key endothelial, dermal, and epidermal genes in 2D culture and HSCs. Epidermal integrity and proliferation were also evaluated through haematoxylin and eosin staining, and IF staining. Results: IF confirmed iEC commitment to endothelial phenotype. RT-qPCR showed HUVECs and iECs immaturity compared with HDBECs. In vitro, the vascular network extension was comparable for HDBECs and HUVECs despite differences in vascular diameter, whereas iECs formed unorganized rudimentary tubular structures. In vivo, all ECs produced discrete vascular networks of varying dimensions. HUVECs and HDBECs maintained a higher proliferation of basal keratinocytes. HDBECs had the best impact on extracellular matrix expression, and epidermal proliferation and differentiation. Innovation: To our knowledge, this study represents the first direct and quantitative comparison of HDBECs, HUVECs, and iECs angiogenic performance in HSCs. Conclusions: Our data indicate that HUVECs and iECs can be an alternative cell source to HDBEC to promote the short-term viability of prevascularized engineered grafts. Nevertheless, HDBECs maintain their capillary identity and outperform other EC types in promoting the maturation of the dermis and epidermis. These intrinsic characteristics of HDBECs may influence the long-term function of skin grafts.

The Effect of Aerobic Training on Tumor Necrosis Factor alpha, Hypoxia-Inducible Factor-1 alpha &amp; Vascular Endothelial Growth Factor Gene Expression in Cardiac Tissue of Diabetic Rats

Objective: The goal of this research was to determine the influence of 4 weeks aerobic training on gene expression of tumor necrosis factor alpha (TNF-α), hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in the cardiac tissue of diabetic rats.&#x0D; Materials and Methods: In an experimental study, 30 male wistar rats were partitioned into three groups (n=10), diabetic aerobic training, diabetic control and healthy control (n=10). Induction of type 2diabetes (T2DM) was done by intraperitoneal infusion of streptozotocin. The progressive aerobic training protocol entailed 4 weeks, 5 sessions per week running on treadmill at velocity of 20 m/min for 60 min. The gene expression of TNF-α, VEGF and HIF-1α were measured by real time &amp; PCR. One way analysis of variance and bonferroni test were applied to analysis the data. The significant level was set at P-value&lt; 0.05.&#x0D; Results: The results indicated that the aerobic training induced significant decrease in TNF-α mRNA (P-value&lt; 0.001) and significant increase in HIF-1α mRNA (P-value&lt; 0.001) and VEGF mRNA (P-value&lt; 0.001) compared to diabetic control group.&#x0D; Conclusion: It appears that aerobic training with reduction of TNF-α issues an affirmative effect on angiogenesis, as a result, it improves diabetic cardiac.

MicroRNAs miR-1 and miR-206 Regulate Monocarboxylate Transporter-4 and Vascular Endothelial Growth Factor Gene Expression in Colorectal Cancer

Background: Colorectal cancer (CRC) is currently the third most common cancer type in males and the second most occurring in females. The role of microRNA (miRNA) in the development of colorectal cancer is not fully elucidated. Therefore, understanding the mechanistic interaction between miRNA and their target oncogenes may hold great importance as a possible target for interventional anticancer therapy.&#x0D; Aims: To identify miRNAs that are part of the regulating pathway of Monocarboxylate Transporter-4 (MCT4) and Vascular Endothelial Growth Factor (VEGF) oncogenes.&#x0D; Study Design: We used publicly available prediction tools (e.g. TargetScan, MicroCosm, PicTar, and DIANA-microT-CDS) to identify the possible miRNA that target the two oncogenes.&#x0D; Methodology: We used the GeneMania database to visualize the network and verify gene names and remove ambiguity and duplications. Furthermore, we used miRTarBase database to identify experimentally validated targets which we used to further confirm miRNA-oncogene relationships. Finally, we utilized miR-Mfold web-tool to further visualize the circular structures and the simulated miR-1 and miR-206 targeting arrangements.&#x0D; Results: We found two putative miRNA (miR-1 and miR-206) that may downregulate MCT4 coded by SLC16A3 gene and VEGF which is coded by VEGF gene. We found relationships between the validated target genes of miR-1 and miR-206 through GeneMania which we extracted from the literature. And we elucidated the proposed structure of these two miRNAs through miR-Mfold web-tool.&#x0D; Conclusion: Our results elucidated a novel regulation pathway in CRC cells and may suggest a potential therapeutic approach for CRC therapy. MiR-1 and miR-206 may help cells go to apoptosis and inhibit the angiogenesis of colorectal cancer cells by down-regulation of MCT4 and VEGF proteins in tumor tissues.

Apoptotic exosome-like vesicles regulate endothelial gene expression, inflammatory signaling, and function through the NF-\u03baB signaling pathway

Persistent endothelial injury promotes maladaptive responses by favoring the release of factors leading to perturbation in vascular homeostasis and tissue architecture. Caspase-3 dependent death of microvascular endothelial cells leads to the release of unique apoptotic exosome-like vesicles (ApoExo). Here, we evaluate the impact of ApoExo on endothelial gene expression and function in the context of a pro-apoptotic stimulus. Endothelial cells exposed to ApoExo differentially express genes involved in cell death, inflammation, differentiation, and cell movement. Endothelial cells exposed to ApoExo showed inhibition of apoptosis, improved wound closure along with reduced angiogenic activity and reduced expression of endothelial markers consistent with the first phase of endothelial-to-mesenchymal transition (endoMT). ApoExo interaction with endothelial cells also led to NF-κB activation. NF-κB is known to participate in endothelial dysfunction in numerous diseases. Silencing NF-κB reversed the anti-apoptotic effect and the pro-migratory state and prevented angiostatic properties and CD31 downregulation in endothelial cells exposed to ApoExo. This study identifies vascular injury-derived extracellular vesicles (ApoExo) as novel drivers of NF-κB activation in endothelial cells and demonstrates the pivotal role of this signaling pathway in coordinating ApoExo-induced functional changes in endothelial cells. Hence, targeting ApoExo-mediated NF-κB activation in endothelial cells opens new avenues to prevent endothelial dysfunction.

Sex diversity in proximal tubule and endothelial gene expression in mice with ischemic acute kidney injury.

Female sex protects against development of acute kidney injury (AKI). While sex hormones may be involved in protection, the role of differential gene expression is unknown. We conducted gene profiling in male and female mice with or without kidney ischemia-reperfusion injury (IRI). Mice underwent bilateral renal pedicle clamping (30 min), and tissues were collected 24 h after reperfusion. RNA-sequencing (RNA-Seq) was performed on proximal tubules (PTs) and kidney endothelial cells. Female mice were resistant to ischemic injury compared with males, determined by plasma creatinine and neutrophil gelatinase-associated lipocalin (NGAL), histologic scores, neutrophil infiltration, and extent of apoptosis. Sham mice had sex-specific gene disparities in PT and endothelium, and male mice showed profound gene dysregulation with ischemia-reperfusion compared with females. After ischemia PTs from females exhibited smaller increases compared with males in injury-associated genes lipocalin-2 (Lcn2), hepatitis A virus cellular receptor 1 (Havcr1), and keratin 18 (Krt18), and no up-regulation of SRY-Box transcription factor 9 (Sox9) or keratin 20 (Krt20). Endothelial up-regulation of adhesion molecules and cytokines/chemokines occurred in males, but not females. Up-regulated genes in male ischemic PTs were linked to tumor necrosis factor (TNF) and Toll-like receptor (TLR) pathways, while female ischemic PTs showed up-regulated genes in pathways related to transport. The data highlight sex-specific gene expression differences in male and female PTs and endothelium before and after ischemic injury that may underlie disparities in susceptibility to AKI.

Covalent Self-Assembly of PEG and Heparin Improves Biological Performance of Electrospun Vascular Grafts for Carotid Artery Replacement

Rapid endothelialization of small-diameter vascular grafts remains a significant challenge in clinical practice. In addition, compliance mismatch causes intimal hyperplasia and finally leads to graft failure. To achieve compliance match and rapid endothelialization, we synthesized low-initial-modulus poly(ester-urethane)urea (PEUU) elastomer and prepared it into electrospun tubular grafts and then functionalized the grafts with poly(ethylene glycol) (PEG) and heparin via covalent self-assembly. The PEG- and heparin-functionalized PEUU (PEUU@PEG-Hep) graft had comparable mechanical properties with the native blood vessel. In vitro data demonstrated that the grafts are of great cytocompatibility and blood compatibility. Covalent self-assembly of PEG and heparin significantly promoted the adhesion, spreading, and proliferation of human umbilical vein endothelial cells (HUVECs) and upregulated the expression of vascular endothelial cell-related genes, as well as increased the capability of grafts in preventing platelet deposition. In vivo assessments indicated good biocompatibility of the PEUU@PEG-Hep graft as it did not induce severe immune responses. Replacement of resected carotid artery with the PEUU@PEG-Hep graft in a rabbit model showed that the graft was capable of rapid endothelialization, initiated vascular remodeling, and maintained patency. This study demonstrates the synergistic effects of compliance match and efficacious antithrombosis of synthetic grafts for blood vessel regeneration.

Abnormal Endothelial Gene Expression Associated With Early Coronary Atherosclerosis.

BackgroundWe examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects.Methods and ResultsSubjects aged <50 years (mean age, 37; range, 22–49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of HMGB1 (high mobility group box 1) which we unexpectedly found to be linked to low LAMC1 (laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio‐functionally. Compared with normal BOEC, abnormal BOEC released 13±3‐fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow‐up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years).ConclusionsThe known pathogenic effects of high‐HMGB1 and low‐LAMC1 predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.

Placental syncytiotrophoblast extracellular vesicles enter primary endothelial cells through clathrin-mediated endocytosis

IntroductionThe aim was to investigate syncytiotrophoblast extracellular vesicle (STBEV) uptake mechanisms by primary endothelial cells, the effects on gene expression, cell activation as well as the effect of aspirin. MethodsThe STBEVs were derived using the placental perfusion system, from normal or preeclamptic placentas. Endothelial uptake was analysed with flow cytometry. To elucidate uptake, different inhibitors were tested; Cytochalasin D, Chlorpromazine hydrochloride, Methyl-B-cyclodextrin, Dynasore and Wortmannin. Endothelial gene expression was evaluated using an endothelial cell biology qPCR array. Cell activation was studied by ICAM-1 surface expression after STBEV exposure, with and without aspirin treatment. ResultsNormal and preeclamptic STBEV uptake was blocked in similar ways. Chlorpromazine, Dynasore and Wortmannin almost completely blocked STBEV uptake. Methyl-B-cyclodextrin blocked 45–60% of the uptake while Cytochalasin D did not block uptake at all. Neither normal nor preeclamptic STBEVs had any significant effects on endothelial gene expression. Normal STBEVs down-regulated cell surface protein ICAM-1 expression, with and without aspirin treatment. Aspirin had no effect on STBEV uptake or cellular gene expression on its own, however it down regulated ICAM-1 protein expression in combination with preeclamptic STBEV exposure. DiscussionSTBEV uptake primarily occurred through clathrin-mediated endocytosis. The STBEVs had no significant effect on gene expression but did have effects on ICAM-1 surface expression. The prophylactic mechanisms of aspirin may be by preventing the endothelium from being activated by the preeclamptic STBEVs.

Uptake of Schistosoma mansoni extracellular vesicles by human endothelial and monocytic cell lines and impact on vascular endothelial cell gene expression

The ability of the parasitic blood fluke Schistosoma mansoni and other parasitic helminths to manipulate host biology is well recognised, but the mechanisms that underpin these phenomena are not well understood. An emerging paradigm is that helminths transfer their biological cargo to host cells by secretion of extracellular vesicles (EVs). Herein, we show that two populations of S. mansoni secreted EVs – exosome-like vesicles (ELVs) and microvesicles (MVs) – are actively internalised in two distinct human cell lines that reflect the resident cell types encountered by the parasite in vivo: human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes. RNA-sequencing of HUVECs co-cultured with S. mansoni ELVs compared with untreated HUVECs revealed differential expression of genes associated with intravascular parasitism, including vascular endothelial contraction, coagulation, arachidonic acid metabolism and immune cell trafficking and signalling. Finally, we show that antibodies raised against recombinant tetraspanin (TSP) proteins from the surface of S. mansoni EVs significantly blocked EV uptake by both HUVECs and THP-1 monocytes whereas pre-immunisation antibodies did not. To our knowledge, this is the first evidence demonstrating the internalisation of secreted EVs from any helminth into vascular endothelial cells, providing novel insight into the potential mechanisms underlying host-schistosome interactions. The ability of anti-TSP antibodies to block vesicle uptake by host target cells further supports the potential of TSPs as promising antigens for an anti-fluke vaccine. It also suggests a potential mechanism whereby the current candidate human schistosomiasis vaccine, Sm-TSP-2, exerts its protective effect in animal models.

Circulating miRNA 887 is differentially expressed in ARDS and modulates endothelial function.

Circulating microRNAs (miRNAs) can be taken up by recipient cells and have been recently associated with the acute respiratory distress syndrome (ARDS). Their role in host predisposition to the syndrome is unknown. The objective of the study was to identify circulating miRNAs associated with the development of sepsis-related ARDS and examine their impact on endothelial cell gene expression and function. We determined miRNA levels in plasma collected from subjects during the first 24 h of admission to a tertiary intensive care unit for sepsis. A miRNA that was differentially expressed between subjects who did and did not develop ARDS was identified and was transfected into human pulmonary microvascular endothelial cells (HPMECs). RNA sequencing, in silico analysis, cytokine expression, and leukocyte migration assays were used to determine the impact of this miRNA on gene expression and cell function. In two cohorts, circulating miR-887-3p levels were elevated in septic patients who developed ARDS compared with those who did not. Transfection of miR-887-3p into HPMECs altered gene expression, including the upregulation of several genes previously associated with ARDS (e.g., CXCL10, CCL5, CX3CL1, VCAM1, CASP1, IL1B, IFNB, and TLR2), and activation of cellular pathways relevant to the response to infection. Functionally, miR-887-3p increased the endothelial release of chemokines and facilitated trans-endothelial leukocyte migration. Circulating miR-887-3p is associated with ARDS in critically ill patients with sepsis. In vitro, miR-887-3p regulates the expression of genes relevant to ARDS and neutrophil tracking. This miRNA may contribute to ARDS pathogenesis and could represent a novel therapeutic target.

Mesenchymal-endothelial transition-derived cells as a potential new regulatory target for cardiac hypertrophy

The role of Mesenchymal-endothelial transition (MEndoT) in cardiac hypertrophy is unclear. To determine the difference between MEndoT-derived and coronary endothelial cells is essential for understanding the revascularizing strategy in cardiac repair. Using lineage tracing we demonstrated that MEndoT-derived cells exhibit highly heterogeneous which were characterized with highly expression of endothelial markers such as vascular endothelial cadherin(VECAD) and occludin but low expression of Tek receptor tyrosine kinase(Tek), isolectin B4, endothelial nitric oxide synthase(eNOS), von Willebrand factor(vWF), and CD31 after cardiac hypertrophy. RNA-sequencing showed altered expression of fibroblast lineage commitment genes in fibroblasts undergoing MEndoT. Compared with fibroblasts, the expression of p53 and most endothelial lineage commitment genes were upregulated in MEndoT-derived cells; however, the further analysis indicated that MEndoT-derived cells may represent an endothelial-like cell sub-population. Loss and gain function study demonstrated that MEndoT-derived cells are substantial sources of neovascularization, which can be manipulated to attenuate cardiac hypertrophy and preserve cardiac function by improving the expression of endothelial markers in MEndoT-derived cells. Moreover, fibroblasts undergoing MEndoT showed significantly upregulated anti-hypertrophic factors and downregulated pro-hypertrophic factors. Therefore MEndoT-derived cells are an endothelial-like cell population that can be regulated to treat cardiac hypertrophy by improving neovascularization and altering the paracrine effect of fibroblasts.

Facio‐genital dysplasia 5 (FGD5) regulates G‐protein coupled receptors (GPCRs) signaling to phosphatidylinositol 3 kinase (PI3K) pathway

IntroductionDrugs targeting the VEGF‐pathway have been approved to treat various cancers. However, tumors become resistant to this therapy due to the recruitment of alternative growth factors, acting via cognate endothelial cells (EC) receptor tyrosine kinases (RTK) or g‐protein coupled receptors (GPCR), to cue neo‐angiogenesis. Facio‐genital dysplasia 5 (FGD5) regulates vascular endothelial growth factor (VEGF)‐mediated angiogenesis, and VEGF receptor‐2 trafficking at the endosome. However, the role of FGD5 to regulate other pro‐angiogenic signaling pathways that co‐operate with VEGF has not been explored. Here, we identify a novel role of FGD5 to control G protein‐coupled receptor (GPCR) signaling.MethodsHuman umbilical vein endothelial cells (HUVEC) were mounted on microcarrier beads, suspended in fibrin gels, then co‐cultured with freshly harvested patient‐derived renal cell carcinoma (PD‐RCC) samples. The effect of FGD5 loss on sprouting angiogenesis was evaluated using RNAi against FGD5 in HUVEC. Stromal cell derived factor (SDF)‐1 was used as a model GPCR ligand to study the role of FGD5 in endothelial angiogenesis and PI3K signaling. Further, FDG5 domain‐deletant mutants were transiently overexpressed to rescue the effect of FGD5 knockdown on endothelial PI3K signaling. Western blot and confocal imaging were performed to readout the role of FGD5 in the GPCR/PI3K signaling pathway.ResultsLoss of FGD5 in HUVEC decreased sprouting angiogenesis stimulated by growth factors released from PD‐RCCs. The defect in angiogenesis was accompanied by downregulation of the expression of endothelial tip cell specific marker genes. Knockdown of endothelial FGD5 abolished the SDF‐1 angiogenic effect and SDF‐1 signaling to PI3K‐β and Akt. Inhibition of Rac1, a Rho GTP‐binding protein required for PI3K‐β activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5 may control PI3K‐β activity through Rac1‐GTP. Overexpression of Dbl (Rho guanine nucleotide exchange factor) domain‐deleted FGD5 (FGD5ΔDbl) reduced SDF‐1‐stimulated Akt phosphorylation, and failed to rescue defective PI3K signalling in FGD5‐deficient cells, indicating that the Dbl domain of FGD5 regulates the activity of PI3K‐β. PI3K‐β and phosphorylated Akt localized to early endosomes after SDF‐1 stimulation, but loss of FGD5 decreased endosomal phosphorylated‐Akt. Failure of Akt activation at PI3K‐β‐positive endosomes suggests a defect in PI3K‐β activity after FGD5 loss.ConclusionsWe identify a novel role of FGD5 to regulate GPCR signaling to PI3K‐β. FGD5 acts at an endosomal convergence node regulating RTK and GPCR angiogenic pathways that may serve as a target for anti‐angiogenic therapy.Support or Funding InformationUniversity of Alberta Doctoral Recruitment Scholarship, Translational Medicine Research Award, Alberta Graduate Excellence Scholarship, Canadian Cancer Society, Division of Nephrology

CCL17 blockade as a therapy for osteoarthritis pain and disease

Purpose: Osteoarthritis (OA) is a major cause of disability and chronic pain has been inadequately managed due to a poor understanding of the underlying mechanisms. We have previously demonstrated a new GM-CSF→Jmjd3→interferon regulatory factor 4 (IRF4)→chemokine (c-c motif) ligand 17 (CCL17) pathway, which is important for the development of inflammatory arthritis pain and disease. Here we investigated the involvement of the pathway in OA pain and disease development using a GM-CSF-dependent collagenase-induced OA model. Methods: CiOA was induced in C57BL/6 wild-type (WT), Irf4-/-, Ccl17E/E, Ccr4-/-, Tnf-/- and GM-CSF-/- mice. Additionally, therapeutic targeting of CCL17, Jmjd3 and cyclooxygenase 2 (COX-2) was evaluated. Development of pain (assessment of weight distribution) and OA disease (histologic scoring of synovitis, cartilage destruction and osteophyte size) were assessed. Synovial joint cells, including neutrophils, macrophages, fibroblasts and endothelial cells, were isolated (cell sorting) and gene expression analyzed (quantitative PCR). Results: Studies in the gene-deficient mice indicated that IRF4, CCL17 and the CCL17 receptor, CCR4, but not TNF, were required for CiOA pain and optimal cartilage destruction and osteophyte size. Therapeutic neutralization of CCL17 and Jmjd3 ameliorated both pain and disease, whereas a COX-2 inhibitor only ameliorated pain. In the synovium, Ccl17 mRNA was only expressed by the macrophages in a GM-CSF- and IRF4-depedent manner. Ccr4 mRNA was expressed by various types of cells, including fibroblasts, while only fibroblasts expressed Mmp3 and Mmp13 in a GM-CSF-, IRF4- and CCL17-dependent manner. Conclusions: The GM-CSF→Jmjd3→IRF4→CCL17 pathway is important for the development of experimental OA pain and disease. Therefore, CCL17 could be a potential therapeutic target for the treatment of both OA pain and disease in patients.