Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo

Summary. Background: Endothelial activation and dysfunction are associated with several diseases. However, hardly any specific markers are available. Microparticles (MP) from endothelial cells (EC; EMP) were reported in patient groups and healthy individuals. The antibodies used to detect EMP, however, were mainly directed against antigens without EC specificity. Objectives: We evaluated the antigens on EC and EMP to establish proper markers for EMP detection. Methods: EMP were isolated from supernatants of resting and interleukin (IL)‐1α activated human umbilical vein EC (HUVEC; n = 3; 0–72 h), stained with annexin V and monoclonal antibodies, and analyzed by flow cytometry. Human platelet‐MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from systemic lupus erythematosus (SLE) patients (n = 11) and healthy individuals (n = 10). Results: Platelet–endothelial cell adhesion molecule‐1 (PECAM‐1), α_ν and β₃ were constitutively exposed on HUVEC, but (almost) absent on EMP (<15% positive for α_ν and β₃), or only exposed on a subpopulation (PECAM‐1; 30–60%). Activated HUVEC (>80%) and (subpopulations of) EMP exposed E‐selectin and tissue factor. PMP strongly exposed PECAM‐1, β₃, and glycoprotein (GP)Ib (CD42b), but not α_ν or E‐selectin. GPIb and P‐selectin (CD62P) were absent on EMP. Plasma samples contained 0.5% MP staining for E‐selectin and/or α_ν. Plasma from one SLE patient contained E‐selectin exposing MP (21%), but little α_ν‐positive MP. Conclusions: EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL‐1α‐stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E‐selectin exposed on IL‐1α‐stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.     

A responsive human triple‐culture model of the air–blood barrier: incorporation of different macrophage phenotypes

Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co‐culture‐systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an anti‐inflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2_wh, wound‐healing; M2_reg, regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established in vitro air–blood barrier co‐culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO‐HAS‐1 on the opposite side of a Transwell filter‐membrane. IL‐8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory‐like response of the air–blood barrier co‐culture, thus demonstrating its potential as a useful in vitro model for inflammatory lung diseases. The two M2 models represent a ‘non‐inflammatory’ phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological in vitro air–blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive in vitro triple‐culture model of the air–blood‐barrier that mimics AM features as they occur in vivo. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.     

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell‐contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC‐lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ₁) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre‐differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre‐differentiated ASCs were used. Pre‐differentiated ASCs had an inferior mitogenic response to EC‐produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co‐culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ₁ induced secretion of activin A. Augmenting co‐culture incubation media with anti‐activin A IgG restored the ability of pre‐differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ₁ or activin A‐induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co‐injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.     

Tissue factor and IL8 production by P-selectin-dependent platelet–monocyte aggregates in whole blood involves phosphorylation of Lyn and is inhibited by IL10

Summary. Background: P‐selectin and CD40L expressed by activated platelets induce tissue factor (TF) and inflammatory cytokines in monocytes, but little is known of the cellular signaling pathways involved. The anti‐inflammatory cytokine IL10 reduces atherosclerotic plaque formation. Objectives: To evaluate the importance of P‐selectin upon platelet–monocyte aggregate (PMA) formation in thrombin receptor activator peptide (TRAP) stimulated whole blood, the P‐selectin–P‐selectin glycoprotein ligand (PSGL)‐1‐induced cellular signaling pathway, and the effects of IL10 on these functions. Methods: TF, IL8, and monocyte chemotactic protein‐1 (MCP‐1) production, PMAs and phosphorylation of Lyn were analyzed in whole blood, purified monocytes, and vitamin D₃‐differentiated U‐937 cells stimulated with TRAP or P‐selectin with or without IL10. Anti‐P‐selectin or anti‐CD40L antibodies (Abs), Src‐kinases inhibitors, SU6656 or PP2, were added in some experiments. Results: TRAP and P‐selectin increased TF, IL8, and MCP‐1 mRNA in whole blood and purified monocytes. Anti‐P‐selectin Ab reduced TRAP‐induced PMA formation by 80 ± 2% (P = 0.001) and production of TF (P = 0.04) and IL8 (P = 0.01). IL10 and SU6656 had no effect on PMA formation, although both significantly reduced TF (P = 0.002 and P = 0.02) and IL8 (P = 0.009 and P = 0.001) mRNA upon TRAP and P‐selectin stimulation. Induced Lyn phosphorylation in monocytes was diminished by SU6656 (P = 0.02), anti‐P‐selectin Ab (P = 0.02), and IL10 (P = 0.03) upon TRAP or P‐selectin stimulation. These results were confirmed in the vitamin D₃‐differentiated U‐937 cells. Conclusions: The formation of PMAs in whole blood was P‐selectin‐dependent in the long term. P‐selectin–PSGL‐1‐induced TF and IL8 expression through Lyn phosphorylation, and part of the inhibitory effect of IL10 depends on reduced phosphorylation.     

Ascidian dermatan sulfates attenuate metastasis,inflammation and thrombosis by inhibition of P‐selectin

See also Zacharski LR. Controlling cancer growth from within the blood coagulation mechanism. This issue, pp 1804–6. Summary. Background: Cancer‐associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti‐inflammatory activities that are linked to inhibition of P‐selectin and/or L‐selectin. P‐selectin‐mediated platelet–tumor cell and tumor cell–endothelium interactions facilitate the initial steps of metastasis. Objectives and Methods: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P‐selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Results: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2‐GalNAc)_n, but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P‐selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC‐38 colon carcinoma and B16‐BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl₃‐induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P‐selectin and thereby the binding of activated platelets during thrombus formation. Conclusions: Collectively, these findings provide evidence that specific inhibition of P‐selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

TLR4 stimulation by LPS enhances angiogenesis in a co‐culture system consisting of primary human osteoblasts and outgrowth endothelial cells

The development of new approaches leading to fast and successful vascularization of tissue‐engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS‐EB) significantly enhances microvessel formation in a co‐culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS‐EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT–PCR have shown that the level of two adhesion molecules (ICAM‐1 and E‐selectin), two cytokines (IL‐6 and IL‐8) and two growth factors (VEGF and PDGF‐BB) related to angiogenesis increase significantly after LPS‐EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

A radiolabeled nonapeptide probe targeting PC‐3 cells and bone metastases of prostate cancer in mice

Previous investigations showed that interleukin‐11 (IL‐11) and the IL‐11 receptor (IL‐11R) are correlated with regulation of tumor progression and may play significant roles in bone metastases. The nonapeptide structure c(CGRRAGGSC) is a phage‐display‐selected IL‐11 mimic that binds to IL‐11R. The aim of this study was to synthesize radiolabeled c(CGRRAGGSC) and to investigate the possible interaction between this radioactive probe and an IL‐11R‐positive bone metastasis model of PC‐3 prostate cancer. The molecular probe ^99mTc–DTPA–c(CGRRAGGSC) was radiolabeled with ^99mTc using the diethylenetriaminepentaacetic acid (DTPA) chelate. Counterstaining was performed with LSS670, a near‐infrared dye. The binding sites of the molecular probe in PC‐3 cells were observed under a fluorescence microscope. The binding characteristics of the labeled probe were analyzed using radioreceptor analysis. Single photon emission tomography imaging and biodistribution of the probe were investigated using xenografts of PC‐3 cells into tibias of nude mice. The labeled product, ^99mTc–DTPA–c(CGRRAGGSC), was obtained with high labeling efficiency, high radiochemical purity and good stability. The molecular probe was combined with the PC‐3 cell membrane and cytoplasm through fluorescence tracing. In the saturation and competitive inhibition experiments performed in vitro, the K_d value was 0.32 ± 0.02 n m and the B_max value was 754 ± 34 fmol mg^?1 pro. The probe exhibited a high tumor uptake in vivo. The radioactive molecular probe ^99mTc–DTPA–c(CGRRAGGSC) may be used as a specific molecular imaging agent for detecting IL‐11R overexpression in tumors and bone metastasis, such as prostate cancers. Copyright © 2012 John Wiley & Sons, Ltd.     

Specific E‐selectin targeting with a superparamagnetic MRI contrast agent

Targeting of the endothelial inflammatory adhesion molecule E‐selectin by magnetic resonance imaging (MRI) was performed with a superparamagnetic contrast agent in the context of in vitro and in vivo models of inflammation. The specific contrast agent was obtained by grafting a synthetic mimetic of sialyl Lewis^x (sLe^x), a natural ligand of E‐selectin expressed on leukocytes, on the dextran coating of ultrasmall particles of iron oxide (USPIO). This new contrast agent, called USPIO‐g‐sLe^x, was tested, in vitro, on cultured human umbilical vein endothelial cells (HUVECs) stimulated to express inflammatory adhesion molecules, and in vivo, on a mouse model of hepatitis. In vitro, HUVECs were stimulated with the pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) and were then incubated with USPIO‐g‐sLe^x or ungrafted USPIO. In vivo, hepatitis was induced on NMRI mice by injection of concanavalin A (Con A). USPIO‐g‐sLe^x and ungrafted USPIO were injected intravenously. In vitro results showed an extensive retention of USPIO‐g‐sLe^x on TNF‐α stimulated HUVECs. Image intensity and R₂ measurements performed on T₂‐weighted MR images demonstrated a significantly higher binding of USPIO‐g‐sLe^x on stimulated HUVECs. In vivo, USPIO are known to pass through the fenestrae of the liver and to be captured by Kupffer cells, inducing a loss of signal intensity on T₂‐weighted MR images. Unexpectedly, when injected to Con A‐treated mice, USPIO‐g‐sLe^x induced a significantly lower attenuation of liver signal intensity than USPIO or USPIO‐g‐sLe^x injected to healthy mice, or USPIO injected to Con A‐treated mice, suggesting that the specific contrast media is retained extracellularly by an interaction with E‐selectin overexpressed on the vascular endothelium. Both in vitro and in vivo results therefore indicate that USPIO‐g‐sLe^x is recognizing endothelial E‐selectin. USPIO‐g‐sLe^x is thus well suited for the MRI diagnosis of inflammation and for the in vitro evaluation of endothelial cells activation. Copyright © 2006 John Wiley & Sons, Ltd.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Inflammatory markers in relation to insulin resistance and the metabolic syndrome

Background Inflammation has repeatedly been demonstrated to be associated with the metabolic syndrome (MetS) and insulin resistance, but the relative importance of different aspects of the inflammatory process is largely unexplored. Design We measured circulating interleukins (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10); other cytokines (tumour necrosis factor‐α, interferon gamma and monocyte chemotactic protein‐1), cell adhesion molecules [vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1, E‐selectin, P‐selectin, l ‐selectin], and systemic inflammation markers [C‐reactive protein (CRP) and leukocyte count] in 943 70 year old participants (50% women) of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study. We related these biomarkers to MetS and the homeostasis model assessment insulin resistance index (HOMA‐IR). Results In a multivariable model including all inflammatory markers conjointly together with sex, log VCAM‐1 [odds ratio (OR), 1·45; 95% confidence interval (CI), 1·22–1·72 per 1 SD increase; P < 0·0001], log E‐selectin (OR, 1·33; 95% CI, 1·12–1·57 per 1SD increase; P = 0·001), and log CRP (OR, 1·41; 95% CI, 1·20–1·66 per 1‐SD increase; P < 0·0001) were independently associated with MetS. These biomarkers were also independently associated with HOMA‐IR. Conclusions Among 17 inflammatory biomarkers, most of them previously not examined in relation to MetS and insulin resistance, VCAM‐1, E‐selectin and CRP demonstrated the strongest associations with MetS and insulin resistance in our community based sample of the elderly. The relative importance of these biomarkers in predicting the development of MetS, insulin resistance and cardiovascular disease needs to be further examined in a longitudinal setting.     

Magnetic resonance molecular imaging of post‐stroke neuroinflammation with a P‐selectin targeted iron oxide nanoparticle

We have developed a magnetic resonance molecular imaging method using a novel iron‐oxide contrast agent targeted towards P‐selectin – MNP‐PBP (magnetic nanoparticle‐P‐selectin binding peptide) – to image endothelial activation following cerebral ischemia/reperfusion. MNP‐PBP consists of ～1000 PBP ligands (primary sequence: GSIQPRPQIHNDGDFEEIPEEYLQ GGSSLVSVLDLEPLDAAWL) conjugated to a 50 nm diameter aminated dextran iron oxide particle. In vitro P‐ and E‐selectin binding was assessed by competition ELISA. Transient focal cerebral ischemia was induced in male C57/BL 6 mice followed by contrast injection (MNP‐PBP; MNP‐NH2; Feridex; MNP‐PBP‐FITC) at 24 h after reperfusion and T₂ magnetic resonance imaging at 9.4 T was performed. Infarction and microvasculature accumulation of contrast agent was assessed in coronal brain sections. MNP‐PBP attenuated antibody binding to P‐selectin by 34.8 ± 1.7%. P‐selectin was preferentially increased in the infarct hemisphere and MNP‐PBP‐FITC accumulation in the infarct hemisphere microvasculature was observed. Compared with the nontargeted iron oxide agents MNP‐NH2 and Feridex, MNP‐PBP showed a significantly greater T₂ effect within the infarction. MR imaging of P‐selectin expression with a targeted iron oxide nanoparticle contrast agent may reveal early endothelial activation in stroke and other neuroinflammatory states. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced leukocyte–platelet cross-talk in Type 1 diabetes mellitus: relationship to microangiopathy

Summary. Background: Platelets and leukocytes may influence each others' function, i.e. platelet–leukocyte cross‐talk. Diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction. Objective: To evaluate platelet–leukocyte cross‐talk, and if this might contribute to platelet and leukocyte dysfunction and microangiopathy in DM patients. Patients and methods: We evaluated platelet and leukocyte function, and cross‐talk between these cells in Type 1 DM patients without (n = 19) and with (n = 20) microangiopathy, and healthy subjects (n = 27), using whole blood flow cytometry. Platelet–leukocyte cross‐talk was studied in hirudinized whole blood incubated at 37 °C with stirring. Results: Basal single platelet P‐selectin and leukocyte CD11b expression were similar in DM patients and healthy subjects, whilst circulating platelet–leukocyte aggregates and plasma elastase levels were elevated in DM patients. The thromboxane A₂ analog U46619 (3 × 10^?7 m ) induced more marked increases of platelet P‐selectin expression and platelet–leukocyte aggregation in DM patients than in healthy subjects. The leukocyte‐specific agonist N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) (10^?7 m ) induced more marked CD11b expression in DM patients with microangiopathy, compared with healthy subjects. Platelet–leukocyte cross‐talk induced by U46619 (10^?6 m ) showed no difference between DM patients and healthy subjects. fMLP (10^?6 m ) evoked marked leukocyte activation, which subsequently caused mild platelet P‐selectin expression. This leukocyte–platelet cross‐talk was more pronounced in DM patients than in healthy subjects. Furthermore, enhanced leukocyte–platelet cross‐talk was correlated to platelet hyperreactivity among DM patients with microangiopathy only. Conclusions: Type 1 DM is associated with platelet and leukocyte hyperactivity, and enhanced leukocyte–platelet cross‐talk, which may contribute to platelet hyperactivity and the microvascular complications seen in Type 1 DM.     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of smoking cessation on markers of inflammation and endothelial cell activation among individuals with high risk for cardiovascular disease

Objective. To prospectively determine the effect of smoking cessation on markers of inflammation and endothelial cell activation. Material and methods: Thirty male and 22 female smokers of >7 cigarettes daily, aged 32–64 years with cardiovascular disease (CVD) or additional risk factors to smoking, participated in a program of smoking cessation with a follow‐up period of 1 year. Cessation was validated by carbon monoxide measurement in expired breath, and 41 of the patients completed the study (17 quitters and 24 non‐quitters). Plasma samples were drawn at baseline and after 1 year, and inflammatory markers were analyzed by enzyme immunoassays. Peripheral blood mononuclear cells (PBMCs) were isolated at baseline and 1 year in 6 quitters and 6 smokers and mRNA levels of interleukin‐8 (IL‐8), tumor necrosis factor α (TNFα) and intercellular adhesion molecule 1 (ICAM‐1) were analyzed by real‐time quantitative RT‐PCR. Results. Our main findings were: (i) While the concentration of soluble (s) ICAM‐1 decreased in quitters, it increased in smokers, with a significant difference in changes between the groups (p = 0.04). (ii) While there was only minor change in mRNA levels of IL‐8 in smokers, those who stopped smoking showed a decrease in the gene expression of IL‐8 (p<0.09; comparing difference in changes). (iii) Concentrations of the other measured parameters (E‐selectin, IL‐6, sCD40 ligand, TNFα, von Willebrand factor, and C‐reactive protein) were unchanged during follow‐up in both groups. Conclusion. Smoking cessation induced a reduction in ICAM‐1, suggesting a novel mechanism for the rapid reduction in the risk of CVD following smoking cessation.     

Exogenous nitric oxide regulates activity and synthesis of vascular endothelial nitric oxide synthase

Background Nitric oxide (NO) – a major signalling molecule of the vascular system – is constitutively produced in endothelial cells (EC) by the endothelial NO synthase (eNOS). Since a reduced NO synthesis is an early sign of endothelial dysfunction and NO delivering drugs are used to substitute the impaired endothelial NO production, we addressed the effect of exogenous NO on eNOS in human umbilical venous endothelial cell cultures. Materials and methods The synthetic NO donor DETA/NO (trade name, but in the following we refer to detNO), that releases NO in a strictly first order reaction with a half life of 20 h, was used in our experiments. Results Short‐term (20–30 min) detNO treatment of EC increases the Ser¹¹⁷⁷ phosphorylation of the constitutively expressed endothelial NOS and the production of endogenous NO generated by eNOS from [³H]arginine. The phosphorylation of eNOS is Akt‐dependent and completely reverted by the phosphatidylinositol‐3 kinase (PI‐3K) inhibitor LY294002. A prolonged continuous exposure of EC to detNO 150 µmol L^?1 over a period of 24–48 h causes a reversible cell cycle arrest at G₁‐phase associated with a larger cell volume and increased cell protein content (hypertrophic phenotype of EC). The eNOS protein and mRNA of the hypertrophic cells and the generation of endogenous NO are reduced but eNOS phosphorylation could still be elevated by stimulation with vascular endothelial growth factor. Conclusions Our data explain clinical studies describing a short‐term but not a long‐term benefit of NO treatment for patients with cardiovascular risk factors. The results could be a rational approach to develop a generation of NO donors accomplishing a retarded release from NO donors that mimic the low continuous pulsatile stress‐induced release of endogenous NO.     

Matrix formation is enhanced in co‐cultures of human meniscus cells with bone marrow stromal cells

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co‐cultured in vitro in three‐dimensional (3D) pellet culture at three different cell–cell ratios for 3 weeks under normal (21% O₂) or low (3% O₂) oxygen tension in the presence of serum‐free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT–PCR. Co‐cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3–1.7‐fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O₂. GAG matrix formation was also enhanced (1.3–1.6‐fold) under 3% O₂ culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG‐rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co‐cultured pellets. However, SOX9 and HIF‐1α mRNA expression were not significantly modulated by co‐culture. Co‐culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co‐delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Serum brain‐derived neurotrophic factor and platelet activation evaluated by soluble P‐selectin and soluble CD‐40‐ligand in patients with acute myocardial infarction

Little is known about the role of neurotrophins (NT) under adult vascular homeostasis in normal and pathological conditions. The NT family, including nerve growth factor and brain‐derived neurotrophic factor (BDNF) are expressed in atherosclerotic vessels. Previous studies demonstrated that plasma BDNF levels were increased in the coronary circulation in patients with unstable angina. However, the role of BDNF during the onset and evolution of unstable angina remains to be elucidated. The objective of this study was to evaluate the relationship between BDNF, functional parameters and biological markers associated with inflammatory processes and platelet activation. BDNF serum levels were assessed in patients with acute myocardial infarction (MI) (n = 20) or stable angina pectoris (SAP) (n = 20) who underwent coronary angiography. Serum levels of IL‐6, MCP1, sVCAM, soluble CD‐40‐ligand (sCD40L) and soluble P‐selectin (sP‐selectin) were measured simultaneously by flux cytometry. Median BDNF levels were higher in the MI than in the SAP group (1730 vs. 877 pg/mL, respectively; P = 0.025). In MI patients, we observed a significant correlation between BDNF and sP‐selectin (r = 0.58, P = 0.023), although we found a non‐significant trend between BDNF and sCD40L (r = +0.35, P = 0.144). By contrast, no such correlation was observed in SAP patients (r = ?0.22, P = 0.425). No difference was observed between the two groups regarding baseline demographics, risk factors, biological data and angiographic findings. The study suggests that BDNF serum levels in MI patients could be related to platelet activation and the inflammatory response. Further studies are needed to investigate the role of NT in the setting of acute MI.