Outside-in Signaling Pathway Research Articles

SARS-CoV-2 spike protein potentiates platelet aggregation via upregulating integrin αIIbβ3 outside-in signaling pathway.

Platelet hyperreactivity is one of the crucial causes of coagulative disorders in patients with COVID-19. Few studies have indicated that integrin αIIbβ3 may be a potential target for spike protein binding to platelets. This study aims to investigate whether spike protein interacts with platelet integrin αIIbβ3 and upregulates outside-in signaling to potentiate platelet aggregation. In this study, we found that spike protein significantly potentiated platelet aggregation induced by different agonists and platelet spreading in vitro. Mechanism studies revealed that spike protein upregulated the outside-in signaling, such as increased thrombin-induced phosphorylation of β3, c-Src. Moreover, using tirofiban to inhibit spike protein binding to αIIbβ3 or using PP2 to block outside-in signaling, we found that the potentiating effect of spike protein on platelet aggregation was abolished. These results demonstrate that SARS-CoV-2 spike protein directly enhances platelet aggregation via integrin αIIbβ3 outside-in signaling, and suggest a potential target for platelet hyperreactivity in patients with COVID-19. HIGHLIGHTS: •Spike protein potentiates platelet aggregation and upregulates αIIbβ3 outside-in signaling. •Spike protein interacts with integrin αIIbβ3 to potentiate platelet aggregation. •Blocking outside-in signaling abolishes the effect of spike protein on platelets.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αIIbβ3 Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αIIbβ3 is essential in hemostasis and thrombosis. In quiescent platelets αIIbβ3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3 outside-in signaling and should not be used interchangeably as readout for αIIbβ3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of αIIbβ3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Abstract 2098: A Novel Mechanism Of Focal Adhesion Kinase Activation By Integrins And Its Role In Outside-in Signaling In Platelets

Ligand binding to integrin αIIbβ3 mediates platelet adhesion/aggregation and transmits outside-in signaling, leading to platelet spreading, granule secretion, and significant expansion of platelet thrombi resulting in thrombosis. Focal Adhesion Kinase (FAK) has been recognized in many cells to play an important role during integrin outside-in signaling. Whereas FAK activation downstream of integrins is characterized by autophosphorylation of FAK at Y 397 followed by Y 576/7 phosphorylation, it is not clear how FAK phosphorylation and activation is regulated and how FAK play roles in outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) plays a key role in integrin-dependent stimulation of FAK phosphorylation and activation and ROS-stimulated FAK activation is downstream of the Gα13-integrin interaction, NOX2 activation, and the Src signaling pathway. Furthermore, FAK, following ROS-dependent activation, is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We further show that FAK is important in amplification of platelet activation and aggregation as well as in vivo thrombosis. Thus, we have identified a novel mechanism of FAK activation and a key role of FAK in integrin outside-in signaling and integrin-dependent ITAM pathway activation, leading to amplification of platelet activation, aggregation, and thrombosis.

JAK2-V617F Promotes up-Regulation of Pro-Inflammatory Cytokines Including IL-1 upon Adhesion-Induced Activation of β1/β2 Integrins

Introduction In JAK2-V617F (JAK2-VF) mutated cells, over-activation of β1/β2 integrin inside-out signaling has been described (Edelmann-Stephan et al., 2017, JCI). Integrins play a fundamental role in cell interaction and cell invasion during inflammatory processes. In patients suffering from Myeloproliferative Neoplasms (MPNs), highly elevated pro-inflammatory cytokine levels including IL-1β play an important role in pathophysiology. Importantly, Rai et al. (2023, BioRχ) have shown that IL-1β promotes initiation of MPN. In this study, we investigated a potential link between integrin over-activation and elevated pro-inflammatory cytokines including IL-1α and IL-1β in JAK2-VF induced disease. Methods We employed (1) murine myeloid progenitor 32D cells co-transfected with EPO-R and JAK2-WT or JAK2-VF, (2) granulocytes and hematopoietic progenitor cell populations from total bone marrow of Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice (described by Mullally et al. 2010), and (3) human granulocytes isolated from PB of JAK2-VF positive patients and healthy volunteers. Cells were allowed to adhere on immobilized VCAM-1/ICAM-1 or on BSA (control) and subsequently subjected to further analysis including (1) qPCR analysis and ELISA of a panel of inflammatory cytokines (IL-1β, IL-1α, TNF-α, IL-6, CXCL10), (2) Western-blotting monitoring integrin outside-in signaling pathways, (3) FLICA assays assessing adhesion-induced active caspase-1, (4) RNAseq of mouse and human granulocytes (3 JAK2-VF patients, 2 controls). Results In the 32D cell model, adhesion on mVCAM-1/mICAM-1 strongly induced mRNA expression of IL-1α, IL1-β, IL-6, TNF-α and CXCL10. Interestingly, cytokine RNA induction was significantly higher in 32D JAK2-VF cells as compared to the 32D JAK2-WT cells (Figure 1). By applying a panel of integrin signaling inhibitors we could show an essential involvement of pivotal signaling nodes such as PI3K, Syk, FAK and NFκB, respectively. Next, cytokine proteins were investigated by ELISA. Adhesion on mVCAM-1/mICAM-1 increased intracellular IL-1α and IL-1β cytokine levels. Again, this effect was strongly amplified by the JAK2-VF mutation (Figure 2). Importantly, both cytokines were found in their mature form, indicating intact protein processing. RNAseq of bone marrow (BM) derived mouse granulocytes (including immature pro-and pre-neutrophils) also showed significant upregulation of a number of pro-inflammatory genes including Nlrp3, Il1a, Il1b, Tnf, Cxcl2 and Cxcl10 upon adhesion on mVCAM-1/mICAM-1. However, no significant differences between WT and VF cells were observed. In contrast, RNAseq of VCAM-1/ICAM-1 stimulated human JAK2-VF positive PB granulocytes (which represent terminally differentiated cells) revealed no cytokine up-regulation. Based on this finding, we hypothesize that the adhesion-induced cytokine phenotype is restricted to more immature cell populations as observed in the 32D cell model. Therefore, we are currently investigating (by qPCR and intracellular IL-1 immunophenotyping) hematopoietic progenitor cell populations from Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice. Since active IL-1β requires processing by the inflammasome, FLICA assays for active caspase-1 were performed using mouse hematopoietic progenitor cells. Importantly, CD41+ cells and megakaryocyte progenitors (MKP) showed an increased FLICA signal upon adhesion on mVCAM-1/mICAM-1. This indicates that caspase 1 is activated upon adhesion in distinct progenitor cell populations. Finally, to support our hypothesis that JAK2-VF augments induction of pro-inflammatory cytokines upon adhesion to VCAM-1/ICAM-1, Vav1-Cre x JAK2 VF/+ mice (n=8) were treated in vivo with anti-β1/β2-integrin antibodies for 1 week. As expected we found a strong reduction of IL-1α concentration in serum of antibody treated mice (528.7 ± 82.2 pg/ml) as compared to PBS-treated control mice (1302.3 ± 370.4 pg/ml, p = 0.0541) Conclusion Here, we show a functional link between elevated cytokine levels and integrin activation in JAK2-VF induced disease. Adhesion to VCAM-1/ICAM-1 promoted the expression of a number of pro-inflammatory cytokines which are known to play important roles in initiation, clinical symptoms and prognosis of MPNs. Further studies aimed at precisely defining the primary cell populations involved in the mouse MPN disease models as well as in humans are currently ongoing.

LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

Roles of focal adhesion proteins in skeleton and diseases.

The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.

Extract of Seaweed Codium fragile Inhibits Integrin αIIbβ3-Induced Outside-in Signaling and Arterial Thrombosis.

Seaweeds are thought to be promising candidates for functional foods and to help prevent thrombotic and related cardiovascular diseases. Codium fragile (Suringer) Hariot has been traditionally used as a culinary ingredient, and it possesses a range of biological activities, including the inhibition of platelet function. However, the mechanism of this inhibition is unclear. The aim of this study was to examine the inhibitory effect of C. fragile in platelet function. The antiplatelet activity of C. fragile on agonist-activated platelet aggregation, granule secretion, calcium mobilization, platelet spreading, and clot retraction was assessed. The phosphorylation of c-Src, Syk, PLCγ2, and several proteins involving in the αIIbβ3 integrin outside-in signaling pathway were also studied in thrombin and CRP-stimulated platelets. The antithrombotic effect was investigated in mice using ferric chloride-induced arterial thrombus formation in vivo. Transection tail bleeding time was used to evaluate whether C. fragile inhibited primary hemostasis. The main components and contents of C. fragile ethanol extract were confirmed by GC-MS analysis. C. fragile significantly impaired agonist-induced platelet aggregation granule secretion, calcium mobilization, platelet spreading, and clot retraction. Biochemical analysis revealed that C. fragile inhibited the agonist-induced activation of c-Src, Syk, and PLCγ2, as well as the phosphorylation of PI3K, AKT, and mitogen-activated protein kinases (MAPKs). The inhibitory effect of C. fragile resulted from an inhibition of platelet αIIbβ3 integrin outside-in signal transduction during cell activation. Oral administration of C. fragile efficiently blocked FeCl3-induced arterial thrombus formation in vivo without prolonging bleeding time. GC-MS analysis revealed that phytol was the main constituent and the total content of isomers was 160.8 mg/kg. Our results demonstrated that C. fragile suppresses not only the inside-out signaling of αIIbβ3 integrin but also outside-in signal transmission. Therefore, C. fragile could be an effective antiplatelet therapeutic candidate.

Near-infrared -triggered release of tirofiban from nanocarriers for the inhibition of platelet integrin αIIbβ3 to decrease early-stage neointima formation.

Platelets play an important role in the early stage of arterial remodeling after injury. Integrin GPIIb/IIIα (αIIbβ3) regulates platelet activation in the inside-out and outside-in signaling pathways. The use of tirofiban, an integrin αIIbβ3 inhibitor, in clinical therapy is limited by its short in vivo circulation time. Herein, a controlled drug-release system was formulated using CuS@mSiO2-PEG core-shell nanoparticles as near-infrared-triggered nanocarriers to release tirofiban on demand. The nanocarriers possessed good colloidal stability and very high loading efficiency for the integrin αIIbβ3 inhibitor (14.5 wt% for tirofiban). Local application of αIIbβ3 antagonist-tirofiban on an injured arterial wall inhibited platelet activation, which was accelerated by laser irradiation. Ex vivo platelet-promoted monocyte transmigration trans-well assays revealed decreased monocyte transmigration after platelet activation was inhibited by tirofiban. Two weeks after the wire-induced injury, the intimal area and cellular content were analyzed. The neointimal area was decreased in ApoE-/- mice with CuS@mSiO2-PEG/tirofiban and laser irradiation-promoted tirofiban release, which had limited the neointima formation. The lesions showed a decreased content of macrophages and smooth muscle cells compared with ApoE-/- mice without tirofiban inhibition. Therefore, the action of platelet-integrin αIIbβ3 in neointima formation after vascular injury was successfully inhibited in vivo through the controlled release of tirofiban using a near-infrared-triggered nanocarrier, leading to the decrease of early-stage neointima formation. This study also emphasizes the role of platelets in vascular remodeling and provides a new target, namely integrin αIIbβ3, for the inhibition of neointimal hyperplasia during vascular inflammation.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation

AbstractIt is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β3−/− platelets or by integrin antagonists. The impaired MV release and PS exposure in β3−/− platelets were rescued by expression of wild-type β3 but not a Gα13 binding–deficient β3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse

The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function.

P08.56 Bacitracin inhibits the migration of U87-MG glioma cells via interferences of the integrin outside-in signaling pathway

P08.56 Bacitracin inhibits the migration of U87-MG glioma cells via interferences of the integrin outside-in signaling pathway

Reinforcement of integrin-mediated T-Lymphocyte adhesion by TNF-induced Inside-out Signaling.

Integrin-mediated leukocyte adhesion to endothelial cells is a crucial step in immunity against pathogens. Whereas the outside-in signaling pathway in response to the pro-inflammatory cytokine tumour necrosis factor (TNF) has already been studied in detail, little knowledge exists about a supposed TNF-mediated inside-out signaling pathway. In contrast to the outside-in signaling pathway, which relies on the TNF-induced upregulation of surface molecules on endothelium, inside-out signaling should also be present in an endothelium-free environment. Using single-cell force spectroscopy, we show here that stimulating Jurkat cells with TNF significantly reinforces their adhesion to fibronectin in a biomimetic in vitro assay for cell-surface contact times of about 1.5 seconds, whereas for larger contact times the effect disappears. Analysis of single-molecule ruptures further demonstrates that TNF strengthens sub-cellular single rupture events at short cell-surface contact times. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signaling in the T-lymphocyte initial adhesion machinery.

Influence of P2Y12 polymorphisms on platelet activity but not ex-vivo antiplatelet effect of ticagrelor in healthy Chinese male subjects.

Activation of platelet implicated a series of signal conduction including outside-in and inside-out related receptor-mediated signaling pathways. Ticagrelor is the first reversible P2Y12 receptor antagonist that exhibits rapid antiplatelet effect. Given that platelet aggregation varies among individuals, genetic polymorphisms in P2Y12 and subsequent signal molecular such as the G-protein beta 3 subunit (GNB3) are supposed to influence the antiplatelet effect of ticagrelor. The aim of this study was to determine whether genetic polymorphisms in P2Y12 and GNB3 genes influence ex-vivo antiplatelet activity of ticagrelor in healthy Chinese subjects. A total of 196 healthy Chinese male individuals were recruited. ADP-induced platelet aggregation was determined by using light transmittance aggregometry at baseline and after incubation of the platelet-rich plasma with 15 and 50 μmol/l ticagrelor, respectively. Nine single-nucleotide polymorphisms (SNPs) in P2Y12 and the GNB3 rs5443 polymorphism were genotyped by PCR-direct sequencing. P2Y12 haplotypes were inferred. Baseline platelet aggregation was increased in carriers of the common alleles of P2Y12 SNPs (rs1907637, rs2046934, and rs6809699) and rs6787801 TC heterozygotes (P < 0.05 for all). Results of the haplotype analyses were consistent with those of the single SNPs. Ticagrelor at both concentrations of 15 and 50 μmol/l decreased ADP-induced platelet aggregation significantly (P < 0.05, respectively). Neither single SNPs nor haplotypes of P2Y12 affected ticagrelor-induced ex-vivo inhibition of platelet aggregation. P2Y12 and GNB3 polymorphisms have no effect on the ex-vivo antiplatelet activity of ticagrelor in healthy Chinese male subjects.

PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling.

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal- or PMA-induced NET formation in human and mouse neutrophils. These compounds do not affect PMA- or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.

Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy

We have recently discovered that soluble extracellular matrix constituents regulate autophagy via an outside-in signaling pathway. Decorin, a secreted proteoglycan, evokes autophagy in endothelial cells and mitophagy in breast carcinoma cells. However, it is not known whether decorin expression can be regulated by autophagic stimuli such as mTOR inhibition or nutrient deprivation. Thus, we tested whether pro-autophagic stimuli could affect decorin expression in mouse cardiac tissue and whether the absence of decorin could disrupt the in vivo autophagic response. We found that nutrient deprivation induced decorin at the mRNA and protein level in vivo and in vitro, a process regulated at the transcriptional level by inhibiting the canonical mTOR pathway. Moreover, Dcn−/− mice displayed an aberrant response to fasting compared to wild-type mice. Our study establishes a new role for an extracellular matrix proteoglycan and provides a mechanistic role for soluble decorin in regulating a fundamental intracellular catabolic process.

Necroinflammation in Kidney Disease.

The bidirectional causality between kidney injury and inflammation remains an area of unexpected discoveries. The last decade unraveled the molecular mechanisms of sterile inflammation, which established danger signaling via pattern recognition receptors as a new concept of kidney injury-related inflammation. In contrast, renal cell necrosis remained considered a passive process executed either by the complement-related membrane attack complex, exotoxins, or cytotoxic T cells. Accumulating data now suggest that renal cell necrosis is a genetically determined and regulated process involving specific outside-in signaling pathways. These findings support a unifying theory in which kidney injury and inflammation are reciprocally enhanced in an autoamplification loop, referred to here as necroinflammation. This integrated concept is of potential clinical importance because it offers numerous innovative molecular targets for limiting kidney injury by blocking cell death, inflammation, or both. Here, the contribution of necroinflammation to AKI is discussed in thrombotic microangiopathies, necrotizing and crescentic GN, acute tubular necrosis, and infective pyelonephritis or sepsis. Potential new avenues are further discussed for abrogating necroinflammation-related kidney injury, and questions and strategies are listed for further exploration in this evolving field.

Sandwich-like Microenvironments to Harness Cell/Material Interactions.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior. This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

Selective serotonin reuptake inhibitors reduce P2Y12 receptor-mediated amplification of platelet aggregation

Selective serotonin reuptake inhibitors (SSRIs) have been reported to reduce platelet aggregation induced by ADP. ADP induces platelet aggregation through two purinergic receptors P2Y1 and P2Y12. We characterized the inhibitory properties of SSRIs on ADP-induced platelet aggregation and investigated the effects of SSRIs on the signaling pathways downstream of P2Y1 and P2Y12 receptors. Specific antagonists were used to evaluate which purinergic receptor-mediated aggregation was influenced by SSRIs. The primary phase of ADP-induced aggregation was not inhibited by citalopram. Citalopram failed to influence ADP-induced platelet shape change, intracellular calcium mobilization and the early phosphorylation of PKCα. Differently, citalopram inhibited the secondary phase of ADP-induced platelet aggregation in a concentration-dependent manner. Other SSRIs, including fluoxetine and sertraline, exhibited the same anti-platelet effects. Under P2Y1 blockade, citalopram inhibited platelet aggregation and integrin αIIbβ3 activation in response to ADP, indicating that citalopram inhibited P2Y12-mediated aggregation. Citalopram concentration-dependently inhibited the phosphorylation of Akt, GSK3β, p38 MAPK and Syk induced by ADP, but showed no effect on the decrease of cAMP and VASP phosphorylation. With integrin αIIbβ3 blockade, however, the phosphorylation of Akt triggered by ADP was unaltered by the addition of citalopram. Taken together, under the stimulation of ADP, SSRIs inhibit the amplification of platelet aggregation secondary to the activation of P2Y12 receptor, and subsequently reduce the activation of the downstream molecules of the outside-in signaling pathways.

The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors

Integrins and other families of cell adhesion receptors are responsible for platelet adhesion and aggregation, which are essential steps for physiological haemostasis, as well as for the development of thrombosis. The modulation of platelet adhesive properties is the result of a complex pattern of inside-out and outside-in signaling pathways, in which the members of the Rap family of small GTPases are bidirectionally involved. This paper focuses on the regulation of the main Rap GTPase expressed in circulating platelets, Rap1b, downstream of adhesion receptors, and summarizes the most recent achievements in the investigation of the function of this protein as regulator of platelet adhesion and thrombus formation.

The roles of integrin β4 in Vascular Endothelial Cells

Integrin heterodimers play diverse and important roles in physiological and pathological processes, such as cell adhesion, migration, proliferation, differentiation, angiogenesis, and tumor progression, via the outside-in and/or inside-out signaling pathways. Aberrant functions of integrins have been implicated in the causation and intervention of multiple diseases. Integrin β(4), a laminin-5 (LN5) receptor, mainly locates in the adhesion structure of hemidesmosome (HD). Most of the previous researches concentrated on the role of integrin β(4) in cancer and cancer therapy, and a few focused on the physiological roles of normal mammalian cells. Recently, accumulating data reveal that integrin β(4) participates in cell death, macroautophagy (hereafter autophagy), senescence, and differentiation regulations in various cell types including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells, and mouse neural cells, implying the key roles of integrin β(4) in the physiological alteration of mammalian cells. Thus, the elucidation of integrin β(4)-mediated signaling may undoubtedly contribute to novel therapeutic strategies for various human diseases, such as vascular and neural disorders. We have reviewed the roles of integrin β(4) in neural cells. In the present review we will discuss the recent research progress in the inherent functions and pharmacological modulation of integrin β(4) in vascular endothelial cells.