Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated ...downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.
•TMT-SPS-MS3 proteomics methods quantify >3000 significant phosphorylation events initiating and progressing platelet GPVI signaling.•Causal analysis infers and organizes >300 signaling relations among established and more novel effectors in platelet GPVI/ITAM responses.
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Inflammation, the primary response of innate immunity, is essential to initiate the calcification process underlying calcific aortic valve disease (CAVD), the most prevalent valvulopathy in Western ...countries. The pathogenesis of CAVD is multifactorial and includes inflammation, hemodynamic factors, fibrosis, and active calcification. In the development of CAVD, both innate and adaptive immune responses are activated, and accumulating evidences show the central role of inflammation in the initiation and propagation phases of the disease, being the function of Toll-like receptors (TLR) particularly relevant. These receptors act as sentinels of the innate immune system by recognizing pattern molecules from both pathogens and host-derived molecules released after tissue damage. TLR mediate inflammation via NF-κB routes within and beyond the immune system, and play a crucial role in the control of infection and the maintenance of tissue homeostasis. This review outlines the current notions about the association between TLR signaling and the ensuing development of inflammation and fibrocalcific remodeling in the pathogenesis of CAVD. Recent data provide new insights into the inflammatory and osteogenic responses underlying the disease and further support the hypothesis that inflammation plays a mechanistic role in the initiation and progression of CAVD. These findings make TLR signaling a potential target for therapeutic intervention in CAVD.
In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, ...and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis.
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•Non-hypoxic activation of HIF-1α via STAT1 interplay in human aortic valve cell.•Robust pro-angiogenic/inflammatory/calcific effects of IFN-γ and crosstalk with LPS.•Stronger induction of ERK/HIF-1α & angiogenic/osteogenic/apoptotic effects in male AVIC.•Janus Kinase/HIF-1α pathways could be potential therapeutic targets for CAVD.
Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal ...transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin α
IIb
β
3
activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE—Calcific aortic valve disease is the most prevalent valvulopathy in Western countries. An unanticipated pathogenetic clue involving IFN (interferon) was disclosed by the finding of ...constitutive type I IFN activity associated with aortic valve calcification in children with the atypical Singleton-Merten syndrome. On this basis, the role of type I IFN on inflammation and calcification in human aortic valve interstitial cells (AVIC) was examined.
APPROACH AND RESULTS—IFN-α was weakly proinflammatory but potentiated lipopolysaccharide-mediated activation of NF (nuclear factor)-κB and the ensuing induction of proinflammatory molecules in human AVIC. Stimulation with IFN-α and in combination with lipopolysaccharide promoted osteoblast-like differentiation characterized by increased osteoblastic gene expression, BMP (bone morphogenetic protein)-2 secretion, and ectopic phosphatase activity. Sex differences were observed. Likewise, IFN-α treatment of human AVICs in osteogenic medium resulted in increased formation of calcific nodules. Strikingly, IFN-α–mediated calcification was significantly higher in AVICs from males, and was blocked by tofacitinib, a JAK (Janus kinase) inhibitor, and by a BMP antagonist. A female-specific protective mechanism involving the activation of PI3K-Akt (protein kinase B) pathways and cell survival was disclosed. Females exhibited higher levels of BCL2 in valve cells and tissues and lower annexin V staining on cell stimulation.
CONCLUSIONS—IFN-α acts as a proinflammatory and pro-osteogenic cytokine in AVICs, its effects being potentiated by lipopolysaccharide. Results also uncovered sex differences with lower responses in female AVICs and sex-specific mechanisms involving apoptosis. Data point to JAK/STAT (signal transducer and activator of transcription) system as a potential therapeutic target for calcific aortic valve disease.
Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and ...tumor necrosis factor (TNF)-α. Our goal was investigating IFNs’ effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α–induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α–mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.
Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors ...(TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin α
β
activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton's-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.
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