Transfection Of Endothelial Cells Research Articles

Non-viral vaccination through cationic guanidium polymer-pDNA polyplex mediated gene transfer.

Vaccination through cellular transfection of nucleotide-based vaccines is a powerful approach to combatting disease. Plasmid DNA (pDNA) vaccines are particularly promising vectors for non-viral immunomodulation that afford high degrees of potency and flexibility. Versatile guanidinium-functionalized poly(oxanorbornene)imide (PONI-Guan) homopolymers were used to facilitate non-disruptive pDNA condensation into discrete polyplexes, enabling efficient in vitro transfection of endothelial cells and HD-11 macrophages. Translation of these vectors for vaccination of white leghorn chickens against Newcastle disease virus (NDV) elicited strong humoral immune responses against the virus. This approach presents a highly versatile method for targeted immunomodulation in vivo, with the potential for translatability as a non-viral vaccine platform.

Reciprocal regulation of endothelial\u2013mesenchymal transition by MAPK7 and EZH2 in intimal hyperplasia and coronary artery disease

Endothelial–mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFβ1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

Butyrate protects endothelial function through PPARδ/miR-181b signaling

Reports of the beneficial roles of butyrate in cardiovascular diseases, such as atherosclerosis and ischemic stroke, are becoming increasingly abundant. However, the mechanisms of its bioactivities remain largely unknown. In this study, we explored the effects of butyrate on endothelial dysfunction and its potential underlying mechanism. In our study, ApoE-/- mice were fed with high-fat diet (HFD) for ten weeks to produce atherosclerosis models and concurrently treated with or without sodium butyrate daily. Thoracic aortas were subsequently isolated from C57BL/6 wild-type (WT), PPARδ-/-, endothelial-specific PPARδ wild-type (EC-specific PPARδ WT) and endothelial-specific PPARδ knockout (EC-specific PPARδ KO) mice were stimulated with interleukin (IL)-1β with or without butyrate ex vivo. Our results demonstrated that butyrate treatment rescued the impaired endothelium-dependent relaxations (EDRs) in thoracic aortas of HFD-fed ApoE-/- mice. Butyrate also rescued impaired EDRs in IL-1β-treated thoracic aorta ring ex vivo. Global and endothelial-specific knockout of PPARδ eliminated the protective effects of butyrate against IL-1β-induced impairment to EDRs. Butyrate abolished IL-1β-induced reactive oxygen species (ROS) production in endothelial cells while the inhibitory effect was incapacitated by genetic deletion of PPARδ or pharmacological inhibition of PPARδ. IL-1β increased NADPH oxidase 2 (NOX2) mRNA and protein expressions in endothelial cells, which were prevented by butyrate treatment, and the effects of butyrate were blunted following pharmacological inhibition of PPARδ. Importantly, butyrate treatment upregulated the miR-181b expression in atherosclerotic aortas and IL-1β-treated endothelial cells. Moreover, transfection of endothelial cells with miR-181b inhibitor abolished the suppressive effects of butyrate on NOX2 expressions and ROS generation in endothelial cells. To conclude, butyrate prevents endothelial dysfunction in atherosclerosis by reducing endothelial NOX2 expression and ROS production via the PPARδ/miR-181b pathway.

Isolation of Primary Human and Rodent Brain Microvascular Endothelial Cells: Culturing, Characterization, and High-Efficiency Transfection.

Studies of blood-brain barrier (BBB) require developing of a novel andconvenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

Endothelial HSPA12B Exerts Protection Against Sepsis-Induced Severe Cardiomyopathy via Suppression of Adhesion Molecule Expression by miR-126.

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells (ECs) and has been reported to protect against cardiac dysfunction from endotoxemia or myocardial infarction. This study investigated the mechanisms by which endothelial HSPA12B protects polymicrobial sepsis–induced cardiomyopathy. Wild-type (WT) and endothelial HSPA12B knockout (HSPA12B–/–) mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP). Cecal ligation and puncture sepsis accelerated mortality and caused severe cardiac dysfunction in HSPA12B–/– mice compared with WT septic mice. The levels of adhesion molecules and the infiltrated immune cells in the myocardium of HSPA12B–/– septic mice were markedly greater than in WT septic mice. The levels of microRNA-126 (miR-126), which targets adhesion molecules, in serum exosomes from HSPA12B–/– septic mice were significantly lower than in WT septic mice. Transfection of ECs with adenovirus expressing HSPA12B significantly increased miR-126 levels. Increased miR-126 levels in ECs prevented LPS-stimulated expression of adhesion molecules. In vivo delivery of miR-126 carried by exosomes into the myocardium of HSPA12B–/– mice significantly attenuated CLP sepsis increased levels of adhesion molecules, and improved CLP sepsis–induced cardiac dysfunction. The data suggest that HSPA12B protects against sepsis-induced severe cardiomyopathy via regulating miR-126 expression which targets adhesion molecules, thus decreasing the accumulation of immune cells in the myocardium.

Early-onset preeclampsia, plasma microRNAs, and endothelial cell function

Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function invitro. We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age-matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether overexpression of the microRNAs in endothelial cells affected endothelial cell function invitro. Functional assays were the wound-healing assay (which measures cell migration and proliferation), the proliferation assay, and the tube-formation assay (which assesses formation of endothelial cell tubes during the angiogenic process). To determine whether the miRNAs are able to decrease gene expression of certain genes, RNA was isolated from transfected endothelial cells and gene expression (by measuring RNA expression) was evaluated by gene expression microarray (Genechip Human Gene 2.1 ST arrays; Life Technologies). For the microarray, we used pooled samples, but the differently expressed genes in the microarray were validated by real-time quantitative polymerase chain reaction in individual samples. No significant differences (fold change <-1.2 or >1.2 with a false-discovery rate <0.05) were found in miRNA plasma concentrations between healthy pregnant and nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy were miR-574-5p, miR-1972, and miR-4793-3p. Transfection of endothelial cells with these miRNAs in showed that miR-574-5p decreased (P<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.1) to decrease both the wound-healing capacity and tube formation invitro. Gene expression analysis of transfected endothelial cells revealed that miR-574-5p tended (P<.1) to decrease the expression of the proliferation marker MKI67. We conclude that in the early-onset preeclampsia group in our study different concentrations of plasma miRNAs are present as compared with healthy pregnancy. Our results suggest that miR-574-5p and miR-1972 decrease the proliferation (probably via decreasing MKI67) and/or migration as well as the tube-formation capacity of endothelial cells. Therefore, these miRNAs may be antiangiogenic factors affecting endothelial cells in preeclampsia.

Abstract 896: Cardiomyocyte-derived Mir-200c-3p In Exosomes Affects Endothelial Angiogenic Capacity And Impairs Cardiac Function

While cardiomyocytes (CMs) have been the main subject of extensive research, the role of other cardiac cell types, such as fibroblasts and endothelial cells (ECs), received considerable less attention in the pathogenesis of heart failure (HF). MiRNAs have recently emerged as mediators of paracrine signaling by being selectively incorporated in exosomes and exchanged between different cell types. The aim of our study is to investigate a potential paracrine miRNA crosstalk between CMs and cardiac ECs and assess the consequences of such miRNA transfer for cardiac vascular remodeling under pathological conditions. We isolated and characterized exosomes from CMs at baseline or after pathological stimulation with phenylephrine and isoproterenol. Although baseline and stressed CMs secrete miRNA-enriched exosomes at similar rates, comparative analysis of extracellular vesicles from both conditions revealed differential miRNA levels, with miR-200c-3p being highly enriched under stress conditions. Direct transfection of ECs with miR-200c-3p precursor molecules or indirect overexpression through transwell co-culture with stimulated CMs leads to diminished angiogenesis reflected by reduced capacity of ECs to proliferate, migrate, and form tubes. This effect was abrogated when we treated CMs with GW4869, an inhibitor of exosomal biogenesis and release. Next, we tested in vivo two doses of specific miR-200c-3p antagomir, Cy3 labelled, to assess specific target of ECs. FACS analysis on cardiac cells derived from the injected mice, confirmed that a low antagomir dose targets ECs whereas, the high dose of antagomir targets all different cardiac cell types. Moreover, when we treated mice subjected transverse aortic constriction (TAC)-induced cardiac pressure overload with miR-200c-3p antagomir, the animals developed a milder hypertrophic phenotype, smaller fibrotic areas, higher amount of capillaries and preserved cardiac ejection fraction, when compared to untreated pressure overloaded mice. Altogether, our results showing exosomal transfer of miR-200c-3p from CMs to ECs indicate the importance of cardiac intercellular communication in the pathophysiology of HF and identify a potential new therapeutic target for intervention strategies.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Multitargeting Peptide-Functionalized Star-Shaped Copolymers with Comblike Structure and a POSS-Core To Effectively Transfect Endothelial Cells.

Gene therapy meets one serious bottleneck in clinical application, namely, lack of safe and efficient gene delivery systems. In order to solve this problem, we designed a lowly cytotoxic and highly efficient gene delivery system for the transfection of endothelial cells. Octa(3-ammoniumpropyl)octasilsesquioxane octachloride reacted with 2-bromoisobutyryl bromide under alkaline condition, and sequentially initiated 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) monomethacrylate (PEGMA) via atom transfer radical polymerization (ATRP) to prepare the eight-arm copolymer with a biocompatible polyhedral oligomericsilsesquioxane (POSS). The side chain ends of the comblike PPEGMA were double-bonded to facilitate the attachment of CAGW or CAG-TAT-NLS functional peptide, thereby enabling the star-shaped copolymers with multifunction. The peptide-functionalized star-shaped copolymers were self-assembled into nanoparticles (NPs) and used to condense pEGFP-ZNF580 (pDNA) to prepare the NPs/pDNA complexes. These complexes had low toxicity as confirmed by MTT. The results of fluorescence microscopy and flow cytometry showed that these multitargeting functionalized gene complexes could effectively transfect endothelial cells. Their transfection efficiency is higher than the positive control PEI 25 kDa group. Moreover, the Western blot test, wound healing assay, and in vitro tube formation assay also demonstrated that the transfected cells showed high migration and enhanced angiogenesis. The pDNA was effective delivered and expressed in endothelial cells by these multitargeting functionalized gene complexes, and promoted cell migration and tube formation. These star-shaped copolymers with comblike structure and a POSS core are a potential gene carrier for gene therapy.

Exenatide exhibits anti-inflammatory properties and modulates endothelial response to tumor necrosis factor α-mediated activation.

Cardiovascular disease is the main cause of mortality and morbidity in the industrialized world. Incretin-mimetic compounds such as exenatide are currently used in the treatment of type 2 diabetes. We investigated the effects of incretin drugs on apoptosis, adhesion molecule expression, and concentration of extracellular matrix (ECM) metalloproteinases under inflammatory conditions within the context of atherosclerotic plaque formation of both human coronary artery endothelial cells (hCAECs) and human aortic endothelial cells (hAoECs). TNF-α-stimulated hCAEC and hAoEC were treated with exenatide (1 and 10nmol/L) and GLP-1 (10 and 100nmol/L) then evaluated for caspase 3/7 activity and assayed for protein levels of adhesion molecules sICAM-1, sVCAM-1, and P-selectin. Concentrations of matrix metalloproteinases (MMPs) MMP-1, MMP-2, MMP-9, and their inhibitors-tissue inhibitor of metalloproteinases (TIMPs), TIMP-1, TIMP-2 were also measured to evaluate the effects on extracellular matrix turnover within an inflammatory environment. Intracellular signaling pathways were evaluated via transfection of endothelial cells with a GFP vector under the NF-κB promoter. Our experimental data suggest that GLP-1 receptor (GLP-1R) agonists downregulate activation of NF-κB and adhesion molecules ICAM and VCAM, but not P-selectin, in both endothelial cell lines. Exendin-4 and GLP-1 modulate the expression of MMPs and TIMPs, with statistically significant effects observed at high concentrations of both incretins. Expressive modulation may be mediated by NF-κB as observed by activation of the vector when stimulated under inflammatory conditions. These findings indicate that GLP-1 analogs have anti-inflammatory properties in endothelial cells that may play an important role in preventing atherosclerosis.

Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells.

In order to improve the transfection efficiency and reduce the cytotoxicity of gene carriers, many strategies have been used to develop novel gene carriers. In this study, five complex micelles (MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k)) were prepared from methoxy-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (mPEG-b-PLGA) and sorbitol-poly(d,l-lactide-co-glycolide)-graft-PEI (sorbitol-PLGA-g-PEI, where the designed molecular weights of PLGA chains were 2 kDa, 4 kDa, 6 kDa, 8 kDa, and 10 kDa, respectively) copolymers by a self-assembly method, and the mass ratio of mPEG-b-PLGA to sorbitol-PLGA-g-PEI was 1/3. These complex micelles and their gene complexes had appropriate sizes and zeta potentials, and pEGFP-ZNF580 (pDNA) could be efficiently internalized into EA.hy926 cells by their gene complexes (MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA). The MTT assay results demonstrated that the gene complexes had low cytotoxicity in vitro. When the hydrophobic PLGA chain increased above 6 kDa, the gene complexes showed higher performance than that prepared from short hydrophobic chains. Moreover, the relative ZNF580 protein expression levels in MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA) groups were 79.6%, 71.2%, and 73%, respectively. These gene complexes could promote the transfection of endothelial cells, while providing important information and insight for the design of new and effective gene carriers to promote the proliferation and migration of endothelial cells.

Peanut arachidin-1 enhances Nrf2-mediated protective mechanisms against TNF-α-induced ICAM-1 expression and NF-κB activation in endothelial cells.

Arachidin-1 [trans-4-(3-methyl-1-butenyl)-3,5,3',4'-tetrahydroxystilbene] is a polyphenol produced by peanut kernels during germination. The aim of the present study was to investigate the mechanism underlying the anti-inflammatory effect of arachidin-1 in endothelial cells (ECs). The results of cell adhesion and western blotting assays demonstrated that arachidin-1 attenuated tumor necrosis factor (TNF)-α-induced monocyte/EC adhesion and intercellular adhesion molecule-1(ICAM-1) expression. Arachidin-1 was demonstrated to exert its inhibitory effects by the attenuation of TNF-α-induced nuclear factor-κB (NF-κB) nuclear translocation and inhibitor of κB-α (IκBα) degradation. Furthermore, arachidin-1 upregulated nuclear factor-E2-related factor-2(Nrf-2), a known mediator of phaseII enzyme expression, and increased the transcriptional activity of antioxidant response element. Transfection of ECs with Nrf-2 siRNA blocked the inhibitory effect of arachidin-1 on ICAM-1 expression, NF-κB nuclear translocation and IκBα degradation. In addition, arachidin-1 induced the expression of the phaseII enzymes thioredoxin-1, thioredoxin reductase-1, heme oxygenase-1, glutamyl-cysteine synthetase and glutathione S-transferase. Following arachidin-1 pretreatment, the H2O2-induced generation of reactive oxygen species was reduced. Therefore, the present results indicate that arachidin-1 suppresses TNF-α-induced inflammation in ECs through the upregulation of Nrf-2-related phaseII enzyme expression.

Double-stranded DNA induces a prothrombotic phenotype in the vascular endothelium

Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.

Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells.

Gene therapy demonstrates promising prospects on cardiovascular diseases. However, nonviral gene delivery system has relatively low transfection efficiency, especially for endothelial cells (ECs). Herein, typical cell-penetrating peptide (TAT), nuclear localization signals (NLSs), and REDV functional peptide have been used to prepare multitargeting complexes. These complexes exhibit higher transfection efficiency owing to the targeting sequences of REDV and NLSs as well as the cell-penetrating function of TAT. The multifunction of the complexes provides high cell uptake, endo/lysosomal escape, and nucleus accumulation of the encapsulated DNA. Thus these multitargeting complexes can provide a potential platform for gene delivery, especially for EC transfection.

MicroRNA-125b Prevents Cardiac Dysfunction in Polymicrobial Sepsis by Targeting TRAF6-Mediated Nuclear Factor κB Activation and p53-Mediated Apoptotic Signaling.

This study examined the effect of microRNA-125b (miR-125b) on sepsis-induced cardiac dysfunction. Mouse hearts were transfected with lentivirus expressing miR-125b (LmiR-125b) 7 days before cecal ligation and puncture (CLP)-induced sepsis. Cardiac function was examined by echocardiography before and 6 hours after CLP (n = 6/group). Survival was monitored following CLP-induced sepsis (n = 12/group). LmiR-125b transfection significantly attenuated cardiac dysfunction due to CLP-induced sepsis. Fractional shortening and ejection fraction values were significantly (P < .05) higher in the LmiR-125b-treated CLP group than in the untreated CLP group. Survival outcome in LmiR-125b-transfected septic mice was markedly improved, compared with mice with CLP-induced sepsis. Transfection of LmiR-125b into the heart significantly suppressed the expression of ICAM-1 and VCAM-1, decreased the accumulation of macrophages and neutrophils in the myocardium, and decreased serum levels of tumor necrosis factor α and interleukin 1β by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated nuclear factor κB (NF-κB) activation. In addition, sepsis-induced myocardial apoptosis was markedly attenuated by LmiR-125b transfection through suppression of p53, Bax, and Bak1 expression. In vitro transfection of endothelial cells with miR-125b mimics attenuate LPS-induced ICAM-1 and VCAM-1 expression by suppressing TRAF6 and NF-κB activation. Increased myocardial miR-125b expression attenuates sepsis-induced cardiac dysfunction and improves survival. miR-125b may be a target for septic cardiomyopathy.

OR12 Integrin β1 partners with HLA class II molecules to mediate endothelial cell (EC) functions

Aim Engagement of cell surface HLA class II (HLA-II) regulates multiple cell functions through activating signal transduction pathways. Class II molecules are heterodimers consisting of two transmembrane glycoproteins with short cytoplasmic domains of 12–15 amino acids that lack kinase activity. We hypothesized that ligation of class II molecules by antibodies (Ab) recruits integrin β1 (ITGB1) into a complex with HLA-II to regulate signal transduction and cell function. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation and protein expression. The interactions between ITGB1 and class II were determined by coimmunoprecipitation (co-IP). BrdU assay was used to measure cell proliferation. Wound healing assay and transmigration assays were applied to determine class II-induced cell migration. Results Class II ligation-activated phosphorylation of Src, Erk and Akt was inhibited by siRNA depletion of ITGB1, but not by ITGB3, B4 and B5 siRNA. Class II-induced EC proliferation and migration were also attenuated by ITGB1 siRNA. Co-IP of class II molecules demonstrated that HLA-II Ab increased ITGB1 expression in the complex. Reverse IP showed that HLA-II was recruited into ITGB1 complex. ITGB1 siRNA decreased complex formation between HLA-II and ITGB1. Inhibiting Src and ERK kinases with PP2 and U0126, respectively, decreased class II-induced ITGB1 phosphorylation at Y783 (Fig). Treatment of EC with PP2 or U0126 reduced complex formation between HLA-II and ITGB1 (Fig) and Talin. Conclusion These results show that HLA-II and ITGB1 cooperate to trigger intracellular signaling in EC and stimulate the proliferation and migration of endothelial cells, which may be important in promoting antibody-mediated rejection (AMR) and transplant vasculopathy. These results provide a mechanistic understanding of the role of ITGB1 and HLA class II in AMR and may lead to the development of novel therapeutic strategies to prevent AMR. Download : Download high-res image (401KB) Download : Download full-size image

Re-Endothelialization Study on Endovascular Stents Seeded by Endothelial Cells through Up- or Downregulation of VEGF.

We studied the effects of gene transfection of endothelial cells with vascular endothelial growth factor (VEGF) on re-endothelialization and inhibition of in-stent restenosis. Transfected endothelial cells (ECs) exposed to different VEGF levels were seeded on a stent surface for evaluation in vitro. VEGF121(++) ECs and VEGF121(--) ECs were established using lentiviral-mediated HUVECs transfection. VEGF RNA transcription level and VEGF protein expression were detected by qPCR, Western blot, and ELISA. Methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, and in vitro HUVEC tube formation assay showed that VEGF overexpression promoted cell proliferation, migration, and endothelial capillary-like tube formation. Downregulation of VEGF expression inhibited these activities. Using a rotational culturing system, cells tightly adhered on the stent surface. Stents seeded with transfected ECs at different VEGF levels were implanted in abdominal aortas of New Zealand white rabbits to study re-endothelialization and inhibition of in-stent restenosis. Stents with cells exposed to excess VEGF expression were almost completely covered with cells after stent implantation for 1 week (w). In the VEGF interference group this process was delayed over 4 w due to RNAi-mediated silencing of VEGF. Cryosectioning after 12 w showed that stents seeded with HUVECs exposed to excess VEGF expression significantly reduced the neointima area and stenosis when compared with bare metal stents and stents from the VEGF interference group. Transgenic HUVECs were not found in tissues of experimental animals. Furthermore, cells from these tissues were similar to those from normal tissue. In conclusion, VEGF-mediated endothelialization was found. Furthermore, ECs exposed to VEGF overexpression reduced neointimal hyperplasia, promoted endothelialization, and reduced in-stent restenosis.

OR37 Antibody ligation of Hla B*44:03 fails to stimulate endothelial cell proliferation due to a failure to recruit integrin beta4

Aim Antibody-mediated rejection (AMR) is an important clinical problem after solid organ transplantation. Recipients producing posttransplant HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Notably, not all patients producing post-transplant Ab develop AMR. We postulated that distinct HLA class I alleles may differ in their capacity to transduce signals. Our previous data showed that HLA-B*44:03 was less efficient than other B locus molecules, including B*44:02, to stimulate signal transduction networks in endothelial cells (EC) despite having a similar binding capacity. We tested the hypothesis that failure of B*44:03 to stimulate signal transduction and EC proliferation was due to its inability to recruit integrin β 4. Method Primary human aortic EC (HAEC) were stimulated with human HLA-B12 allele specific mAb, and phosphorylation of signaling proteins was detected by Western Blot. For protein:protein complex formation, HAEC were stimulated with F(ab’)2 fragment of anti-HLA class I (HLA I) mAb W6/32 or mouse IgG as a control. Cell lysates were immunoprecipitated with a mouse anti-HLA-B12 mAb recognizing both B*44:02 and B*44:03 alleles and complex formation between class I and integrin β 4 was detected by Western Blot. Gene silencing was determined with siRNA transfection. EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Treatment of HAEC with anti-B12 mAb stimulated a increase in phosphorylation of Src, mTOR, ERK and Akt in B*44:02, but not in B*44:03 HAEC. Co-immunoprecipitation experiments showed complex formation between HLA I and integrin β 4 in B*44:02 HAEC, but not in HAEC carrying B*44:03. Furthermore, transfection of EC with integrin β 4 siRNA inhibited HLA-B12 mAb-induced protein phosphorylation and cell proliferation. Conclusions Our results demonstrate that HAEC carrying B*44:03 fail to transduce proliferative signals because they are unable to recruit integrin β 4 into a signaling complex. Disrupting complex formation between integrin β 4 and HLA I may provide a new therapeutic target to prevent AMR.

Abstract 662: X-box Binding Protein 1 Splicing Promotes Wound Healing via Modulating Endothelial Nitric Oxide Synthase

Instruction: X-box binding protein 1 (XBP1) is a pivotal transcription factor in the pathogenesis of the immune system and in the endoplasmic reticulum stress response. Our previous studies have proved that XBP1 is involved in VEGF-mediated endothelial cell (EC) proliferation and angiogenesis. However, whether XBP1 can influence EC migration is unknown. Hypothesis: We assessed the hypothesis that XBP1 splicing is involved in endothelial migration contributing to wound healing and angiogenesis. Method and results: In this study, we used EC monolayer wound healing, tube formation and transwell migration models to study the involvement of XBP1 splicing in EC migration. We found that scratching on EC monolayer triggered XBP1 splicing, which was attenuated by the presence of LY294002, FAK-14 and L-NAME, suggesting that PI3K/Akt, FAK and eNOS may be involved in scratching-mediated upregulation of XBP1 splicing. Over-expression of spliced XBP1 (XBP1s) via Ad-XBP1s gene transfer increased EC migration, while knockdown of IRE1α or XBP1 by ShRNA lentiviruses suppressed EC migration. Over-expression of XBP1s upregulated eNOS at mRNA and protein levels leading to the increase of nitric oxide production. The eNOS promoter reporter analysis revealed that XBP1s down-regulated eNOS transcription. The pSI-Check2-eNOS-3’UTR reporter analysis indicated that XBP1s might upregulate eNOS mRNA via 3’UTR-mediated mRNA stabilization. The microRNA array assays found that XBP1s down-regulated microRNAs mir-24, mir-125 and mir-214. Transfection of EC with the miRNA mimics of those three miRNAs attenuated XBP1s-induced eNOS mRNA upregulation and EC migration. Conclusion: These results suggest that XBP1 splicing may increase eNOS mRNA stabilization and nitric oxide production via down-regulating miRNAs that target eNOS mRNA 3’-UTR, leading to EC migration and contributing to wound healing and angiogenesis. Significance: XBP1 splicing may provide a new therapeutic strategy to tackle endothelium injury-related cardiovascular disease.