Flow mediated vasodilation and circulating concentrations of high sensitive C‐reactive protein,interleukin‐6 and tumor necrosis factor‐α in normal pregnancy – The Cardiovascular Risk in Young Finns Study

Background: Traditional risk factors such as hyperlipidemia induce a state of inflammation that impairs vascular function. Despite marked maternal hyperlipidemia, endothelial function improves during pregnancy. In non‐pregnant state increased circulating levels of pro‐inflammatory cytokines and high sensitive C‐reactive protein (hsCRP) lead to attenuated flow mediated vasodilation. Relation between endothelial function and pro‐inflammatory cytokines has not been studied thoroughly in pregnancy. The aim of this study was to evaluate the effect of pregnancy on hsCRP and pro‐inflammatory cytokines and their associations with vascular endothelial function. Methods: As part of population‐based, prospective cohort Cardiovascular Risk in Young Finns study conducted in Finland we measured brachial artery flow mediated dilation (FMD) and serum concentrations of hsCRP, interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) in 57 pregnant Finnish women throughout gestation and 62 control women matched for age and smoking. Results: HsCRP‐concentration was greater in pregnancy compared to non‐pregnant controls (median hsCRP 2·52 mg l^?1 versus 1·21 mg l^?1, P<0·001). IL‐6‐concentration was slightly increased in pregnancy compared with the non‐pregnant controls (median 1·66 versus 1·32 mg l^?1, non‐significant [NS]) and TNF‐α‐concentration was slightly decreased in pregnant group (2·11 versus 2·38 pg ml^?1, NS). FMD increased during pregnancy and IL‐6 had a positive correlation to the FMD in pregnancy (R = 0·288, P = 0·031). Conclusions: Improvement of FMD in normal pregnancy was not affected by increase in hsCRP concentration. We found an association with IL‐6 and FMD but we believe that improvement in endothelial function during normal pregnancy is not caused by variation in hsCRP, IL‐6 or TNF‐α.     

The calcification potential of human MSCs can be enhanced by interleukin‐1β in osteogenic medium

Inflammation is one of the key regulators of the repair process in bone tissues. Current data about the effect of interleukin‐1β (IL‐1β) on MSCs and osteoblasts are conflicting. We investigated the long‐term effect of IL‐1β on direct osteogenic differentiation of hMSCs in vitro. IL‐1β‐stimulated cells showed enhanced proliferation and entered maturation prior to non‐stimulated ones, as monitored by ALP activity. The process of calcification was accelerated during long‐term stimulation of hMSCs with IL‐1β. Since donor variability is a well‐known issue, we suggest a new method to illustrate global changes of a random chosen donor population through collative analysis. We further demonstrate an absorbance assay to evaluate the degree of calcification during in vitro culture of monolayer expanded hMSCs. Our findings support the importance of IL‐1β in osteogenic differentiation of hMSCs in an in vitro monolayer culture model. A new online absorbance assay is a useful method to evaluate the osteogenic differentiation of hMSCs at early stages. These findings will be helpful in optimizing predifferentiation of hMSCs in vitro for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

Neuropharmacological effects of lipoic acid and ubiquinone on the mRNA level of interleukin‐1β and acetylcholinesterase activity in rat hippocampus after seizures

The purpose of this study was to investigate the neuroprotective effects of lipoic acid and ubiquinone on interleukin‐1β (IL‐1β) mRNA levels and acetylcholinesterase (AChE) activities in rat hippocampus after pilocarpine‐induced seizures. Wistar rats were intraperitoneally administered with either 0.9% saline (icontrol group), LA (10 or 20 mg/kg, LA10 or LA20 groups), UQ (20 or 40 mg/kg, UQ20 and UQ40 groups), pilocarpine (400 mg/kg, P400 group), or co‐administration of pilocarpine with LA or UQ groups 30 min prior to LA or UQ administration. After the treatments, all groups were observed for 1 h. IL‐1β mRNA and AChE activity in rat hippocampus at 1 h after SE onset was determined. Results showed that rats pretreated with LA or UQ developed less seizures and SE more slowly and has less number than animals treated with pilocarpine alone. Reduced IL‐1β mRNA and marked AChE activities in the hippocampus were significantly higher in rats pretreated with LA or UQ in comparison with the values of the control and seized groups. Our findings strongly support the hypothesis that an increase on IL‐1β mRNA levels in hippocampus occurs during seizures induced by pilocarpine, which indicates that inflammatory process plays a crucial role in seizures pathogenic consequences. Our result also suggests that LA or UQ can exert significant neuroprotective effects, at least in part, because of the increase in the AChE activities in rat hippocampus that will be useful in the treatment of neurodegenerative diseases.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

Complement receptor 3 (CD11b/CD18) is implicated in the elimination of β‐amyloid peptides

Microglia are the professional phagocytes of the brain and express phagocytic receptors such as complement receptor 3 (CR3 or CD11b/CD18). Using mimics of the amyloid deposit made of heat‐killed yeasts coated with either Aβ 1‐40 or Aβ 1‐42, we were able to study how microglia interacted with and ingested these particles in vitro. We have shown previously that the low density lipoprotein receptor‐related protein (LRP) is largely implied in the phagocytosis of Aβ 1‐42‐opsonized heat‐killed yeasts and partly in that of Aβ 1‐40‐opsonized heat‐killed yeasts. Here, we report that antibodies against CD11b or CD18 reduced the uptake of the artificial amyloid deposit by microglial cell showing that CR3 is involved in the mechanism. Moreover, a concomitant inhibition of LRP and CR3 completely blocked the ingestion of both kinds of particles suggesting that no other receptors participate to this mechanism.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Plasma (1→3)-β-D-glucan assay and immunohistochemical staining of (1→3)-β-D-glucan in the fungal cell walls using a novel horseshoe crab protein (T-GBP) that specifically binds to (1→3)-β-D-glucan

A highly sensitive enzyme-linked immunosorbent assay specific to (1→3)-β-D-glucans (GBP-ELISA) has been developed using a novel (1→3)-β-D-glucan-binding protein (T-GBP), which was purified from the amebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. This method allowed quantitation of the glucans in a concentration range of 0.1–1,000 ng/ml, regardless of linear and branched structures, and was applied to determine the amounts of (1→3)-β-D-glucan in human and animal plasmas for diagnosis of fungemia. High levels of plasma glucan contents in clinical samples were found to be correlated closely with the severity of fungal infection. T-GBP was successfully utilized for indirect immunofluorescence staining of (1→3)-β-D-glucan in Candida albicans cell walls. J. Clin. Lab. Anal. 11:104–109. © 1997 Wiley-Liss, Inc.