Global Transcriptional Profiling Reveals Novel Autocrine Functions of Interleukin 6 in Human Vascular Endothelial Cells.

BACKGROUND: Interleukin 6 (IL6) is a multifunctional cytokine produced by various cells, including vascular endothelial cells. IL6 has both pro- and non-/anti-inflammatory functions, and the response to IL6 is dependent on whether it acts via the membrane-bound IL6 receptor α (IL6Rα) (classic signaling) or the soluble form of the receptor (transsignaling). As human endothelial cells produce IL6 and at the same time express IL6Rα, we hypothesized that IL6 may have autocrine functions. METHODS: Knockdown of IL6 in cultured human endothelial cells was performed using siRNA. Knockdown efficiency was evaluated using ELISA. RNA sequencing was employed to characterize the transcriptional consequence of IL6 knockdown, and Ingenuity Pathway Analysis was used to further explore the functional roles of IL6. RESULTS: Knockdown of IL6 in cultured endothelial cells resulted in a 84-92% reduction in the release of IL6. Knockdown of IL6 resulted in dramatic changes in transcriptional pattern; knockdown of IL6 in the absence of soluble IL6Rα (sIL6Rα) led to differential regulation of 1915 genes, and knockdown of IL6 in the presence of sIL6Rα led to differential regulation of 1967 genes (fold change 1.5, false discovery rate < 0.05). Pathway analysis revealed that the autocrine functions of IL6 in human endothelial cells are mainly related to basal cellular functions such as regulation of cell cycle, signaling, and cellular movement. Furthermore, we found that knockdown of IL6 activates functions related to adhesion, binding, and interaction of endothelial cells, which seem to be mediated mainly via STAT3. CONCLUSION: In this study, a large number of novel genes that are under autocrine regulation by IL6 in human endothelial cells were identified. Overall, our data indicate that IL6 acts in an autocrine manner to regulate basal cellular functions, such as cell cycle regulation, signaling, and cellular movement, and suggests that the autocrine functions of IL6 in human endothelial cells are mediated via IL6 classic signaling.

Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation.

The healthy vascular endothelium constantly releases autacoids which cause an increase of intracellular cyclic nucleotides to tame platelets from inappropriate activation. Elevating cGMP and cAMP, in line with previous reports, cooperated in the inhibition of isolated human platelet intracellular calcium-mobilization, dense granules secretion, and aggregation provoked by thrombin. Further, platelet alpha granules secretion and, most relevant, integrin αIIaß3 activation in response to thrombin are shown to be prominently affected by the combined elevation of cGMP and cAMP. Since stress-related sympathetic nervous activity is associated with an increase in thrombotic events, we investigated the impact of epinephrine in this setting. We found that the assessed signalling events and functional consequences were to various extents restored by epinephrine, resulting in full and sustained aggregation of isolated platelets. The restoring effects of epinephrine were abolished by either interfering with intracellular calcium-elevation or with PI3-K signalling. Finally, we show that in our experimental setting epinephrine likewise reconstitutes platelet aggregation in heparinized whole blood, which may indicate that this mechanism could also apply in vivo.

Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα.

Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

Individual variations in platelet reactivity towards ADP, epinephrine, collagen and nitric oxide, and the association to arterial function in young, healthy adults.

INTRODUCTION: Platelet aggregation and secretion can be induced by a large number of endogenous activators, such as collagen, adenosine diphosphate (ADP) and epinephrine. Conversely, the blood vessel endothelium constitutively release platelet inhibitors including nitric oxide (NO) and prostacyclin. NO and prostacyclin are also well-known vasodilators and contribute to alterations in local blood flow and systemic blood pressure. MATERIALS AND METHODS: In this study we investigated individual variations in platelet reactivity and arterial functions including blood pressure and flow-mediated vasodilation (FMD) in 43 young, healthy individuals participating in the Lifestyle, Biomarkers and Atherosclerosis (LBA) study. Platelet aggregation and dense granule secretion were measured simultaneously by light transmission and luminescence. FMD was measured with ultrasound. RESULTS: The platelet function assay showed inter-individual differences in platelet reactivity. Specifically, a sub-group of individuals had platelets with an increased response to low concentrations of ADP and epinephrine, but not collagen. When the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) was combined with high doses of these platelet activators, the results indicated for sub-groups of NO-sensitive and NO-insensitive platelets. The individuals with NO-sensitive platelets in response to SNAP in combination with collagen had a higher capacity of FMD of the arteria brachialis. CONCLUSIONS: Platelet reactivity towards ADP, epinephrine and NO differs between young, healthy individuals. Some individuals have a more effective response towards NO, both in the aspect of platelet inhibition ex vivo, as well as vasodilation in vivo.

Activation of the JAK/STAT3 and PI3K/AKT pathways are crucial for IL-6 trans-signaling-mediated pro-inflammatory response in human vascular endothelial cells.

BACKGROUND: IL-6 classic signaling is linked to anti-inflammatory functions while the trans-signaling is associated with pro-inflammatory responses. Classic signaling is induced via membrane-bound IL-6 receptor (IL-6R) whereas trans-signaling requires prior binding of IL-6 to the soluble IL-6R. In both cases, association with the signal transducing gp130 receptor is compulsory. However, differences in the downstream signaling mechanisms of IL-6 classic- versus trans-signaling remains largely elusive. METHODS: In this study, we used flow cytometry, quantitative PCR, ELISA and immuno-blotting techniques to investigate IL-6 classic and trans-signaling mechanisms in Human Umbilical Vein Endothelial Cells (HUVECs). RESULTS: We show that both IL-6R and gp130 are expressed on the surface of human vascular endothelial cells, and that the expression is affected by pro-inflammatory stimuli. In contrast to IL-6 classic signaling, IL-6 trans-signaling induces the release of the pro-inflammatory chemokine Monocyte Chemoattractant Protein-1 (MCP-1) from human vascular endothelial cells. In addition, we reveal that the classic signaling induces activation of the JAK/STAT3 pathway while trans-signaling also activates the PI3K/AKT and the MEK/ERK pathways. Furthermore, we demonstrate that MCP-1 induction by IL-6 trans-signaling requires simultaneous activation of the JAK/STAT3 and PI3K/AKT pathways. CONCLUSIONS: Collectively, our study reports molecular differences in IL-6 classic- and trans-signaling in human vascular endothelial cells; and elucidates the pathways which mediate MCP-1 induction by IL-6 trans-signaling.

Fucoidan-Mimetic Glycopolymers as Tools for Studying Molecular and Cellular Responses in Human Blood Platelets.

The marine sulfated polysaccharide fucoidan displays superior ability to induce platelet aggregation compared to other sulfated polysaccharides. As such, it is an attractive tool for studying molecular and cellular responses in activated platelets. The heterogeneous structure, however, poses a problem in such applications. This study describes the synthesis of sulfated α-l-fucoside-pendant poly(methacryl amides) with homogeneous structures. By using both thiol-mediated chain transfer and reversible addition-fragmentation chain transfer polymerization techniques, glycopolymers with different chain lengths are obtained. These glycopolymers show platelet aggregation response and surface changes similar to those of fucoidan, and cause platelet activation through intracellular signaling as shown by extensive protein tyrosine phosphorylation. As the platelet activating properties of the glycopolymers strongly mimic those of fucoidan, this study concludes these fucoidan-mimetic glycopolymers are unique tools for studying molecular and cellular responses in human blood platelets.

Platelets contribute to amyloid-ß aggregation in cerebral vessels through integrin αIIbß3-induced outside-in signaling and clusterin release.

Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-ß (Aß) in the walls of cerebral vessels. CAA and Aß deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aß deposits, to CAA. We found that synthetic monomeric Aß40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbß3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aß aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbß3 activation, further increasing the secretion of clusterin and Aß40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbß3, indicated that the abundance of this integrin dictated Aß-induced clusterin release and platelet-induced Aß aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aß aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aß aggregates and that Aß, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.

The toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade.

The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARs, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLCγ2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI. Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking ß(3), in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin ß(3) signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLCγ2.

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity.

Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets.

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²âº mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

Characterization of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes.

Platelets are anucleate blood cells that play a crucial role in thrombosis and hemostasis. Despite their lack of nuclear DNA, platelets contain significant amounts of microRNA (miRNA) that may have vital functions in post-transcriptional gene regulation. Here, we combined comprehensive miRNA expression profiling by quantitative PCR with target prediction analysis for the most abundant miRNAs in human platelets. A network composed of predicted platelet miRNA target genes was then constructed, using annotations available in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In addition, we evaluated possible differences in miRNA levels between resting and thrombin-stimulated platelets. We identified 281 transcripts, including 228 mature miRNAs and 53 minor miRNAs (or miR*), of which six miRNAs (miR-15 a, miR-339-3 p, miR-365, miR-495, miR-98, and miR-361-3 p) were up- or down-regulated in activated human platelets (P ≤ 0.001). A redundancy-reduced network was established that encompassed 246 genes in five statistically significant functional clusters representing platelet miRNA regulating pathways. Comparison of the 246 network genes with the platelet mRNA expression data available at ArrayExpress database confirmed that most of these genes (89%) are expressed in human platelets. In conclusion, this work affirms a recent microarray study reporting a wide-spread existence of miRNAs in human platelets. Further, we observed that thrombin stimulation was associated with altered levels of some miRNAs in platelets. The proposed functional network, combining computational prediction analysis with annotations from experimental observations, may in addition provide some information about probable miRNA target pathways in human platelets.

P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets.

In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi(2)-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin alpha(IIb)beta(3) outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA(2)-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA(2) receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA(2) signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled alpha(2A)-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI(2) or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.

ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets.

Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.