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.     

E‐selectin gene in essential hypertension: a case‐control study

Background

Hypertension is associated with endothelial cell dysfunction. E‐selectin, an endothelial cell adhesion molecule, is specific for endothelial cell activation. Polymorphism in E‐selectin gene has recently been identified among which Leu554Phe E‐selectin gene polymorphism is least investigated in essential hypertension. This study reports the association of E‐selectin gene Leu554Phe polymorphism and the expression of E‐selectin gene in patients with essential hypertension.

Materials and methods

We analysed the Leu554Phe polymorphism and expression of E‐selectin gene in 250 patients with essential hypertension and 250 normal healthy controls. Genotyping of Leu554Phe polymorphism was performed by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), and the expression of E‐selectin gene at mRNA and protein levels were carried out by real‐time PCR and Western blot, respectively.

Results

A significant association of E‐selectin genotypes (CT + TT) with essential hypertension (P < .0001, Odds ratio = 2.2 [1.58‐3.24] at 95% CI) was observed. The expression of mRNA for E‐selectin gene in patients with essential hypertension was ~12‐fold higher as compared to control. We observed an elevated level of E‐selectin protein expression (up to 1.9 times) in patients as compared to controls.

Conclusions

A significant association of E‐selectin (Leu554Phe) gene and increased expression of E‐selectin gene at mRNA and protein levels in patients might be related to the genetic predisposition to develop essential hypertension.     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

Synergic effects of VEGF‐A and SDF‐1 on the angiogenic properties of endothelial progenitor cells

Here we investigated the impact of hypoxic environment on the angiogenic properties of early‐outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor‐A (VEGF‐A) and stromal derived factor‐1 (SDF‐1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF‐A did not change the ability of EPC‐conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro‐angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF‐A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF‐depleted EPC‐CM sustained tissue perfusion at the same level as complete EPC‐CM. However, concomitant deletion of VEGF‐A and SDF‐1 in EPC‐CM impaired the pro‐angiogenic properties of EPC‐CM, by inhibition of EC spreading in culture, tube‐like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF‐A or SDF‐1 from EPC‐CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF‐A and SDF‐1 from EPC‐CM impairs its pro‐angiogenic effect, both in vitro and in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Phosphoinositide 3‐kinase mediates CD40 ligand‐induced oxidative stress and endothelial dysfunction via Rac1 and NADPH oxidase 2

Summary. Objectives: CD40 ligand (CD40L) has been implicated as an inducer of reactive oxygen species (ROS) generation in endothelial cells, but definitive evidence for this and the in vivo relevance haves not been demonstrated fully. We thus investigated whether phosphoinositide 3‐kinase (PI3K) was linked to ROS generation and endothelial reactivity in response to CD40L. Methods and Results: CD40L treatment activated PI3K activity by regulating the association between PI3K p85 and the CD40 receptor. CD40L exposure also stimulated the GTPase Rac1, which is known to activate NADPH oxidases, and enhanced ROS formation, whereas PI3K inhibition or depletion by small interfering RNA (siRNA) prevented these responses. Subsequently, PI3K overexpression activated Rac1 and increased ROS generation. These responses were not observed in the presence of inactive Rac1 or siRNA against the NADPH oxidase subunit NOX2. Protein kinase Cζ mediates PI3K‐regulated NADPH oxidase activation by promoting cellular p47phox translocation. Importantly, PI3K inhibition prevented CD40L‐mediated ROS generation and endothelial dysfunction in a mouse model. In summary, PI3K mediates CD40L‐induced ROS production and subsequent endothelial dysfunction. Conclusions: Targeting PI3K may provide a new therapeutic approach in diseases associated with oxidative stress and endothelial dysfunction.     

Elevated plasma levels of P‐selectin glycoprotein ligand‐1‐positive microvesicles in patients with unprovoked venous thromboembolism

Essentials

• PSGL‐1⁺ microvesicles (MVs) may be important in venous thromboembolism (VTE).
• We measured plasma levels and parental origin of PSGL‐1⁺ MVs in patients with unprovoked VTE.
• VTE patients had higher plasma levels of PSGL‐1⁺ MVs than healthy controls.
• The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells.

Summary

Background

Microvesicles (MVs) express antigens from their parental cells and have a highly procoagulant surface. Animal studies suggest that P‐selectin glycoprotein ligand‐1‐positive (PSGL‐1⁺) MVs play a role in the pathogenesis of venous thromboembolism (VTE).

Objective

The aim of this study was to determine plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs in patients with unprovoked VTE.

Methods

We conducted a population‐based case–control study in 20 patients with a history of unprovoked VTE and 20 age‐ and sex‐matched healthy controls recruited from the general population. Plasma levels, the cellular origin and the morphological characteristics of PSGL‐1⁺ MVs were evaluated using flow cytometry, electron microscopy and confocal microscopy.

Results

Plasma levels of PSGL‐1⁺ MVs were associated with increased risk of VTE. The odds ratio per one standard deviation increase in PSGL‐1⁺ MVs was 3.11 (95% confidence interval [CI], 1.41–6.88) after adjustment for age and sex, and 2.88 (95% CI, 1.29–6.41) after further adjustment for body mass index. The PSGL‐1⁺ MVs originated mainly from monocytes and endothelial cells determined by double staining with markers of parental cells using flow cytometry and transmission electron microscopy. Scanning electron microscopy of PSGL‐1‐labeled plasma‐derived MVs displayed dominantly spherical vesicles that varied between 50 and 300 nm in diameter.

Conclusions

Increased plasma levels of PSGL‐1⁺ MVs are associated with the risk of unprovoked VTE. Large population‐based prospective studies are required to validate our findings.

     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y₁ receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm . The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A₂, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y₁₂ receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A₂ generation, ATP release, and P2Y₁₂ activation.     

Effect of chemical immobilization of SDF‐1α into muscle‐derived scaffolds on angiogenesis and muscle progenitor recruitment

The availability of three‐dimensional bioactive scaffolds with enhanced angiogenic capacity that have the capability to recruit tissue specific resident progenitors is of great importance for the regeneration of impaired skeletal muscle. Here, we have investigated whether introduction of chemoattractant factors to tissue specific extracellular matrix promotes cellular behaviour in vitro as well as muscle progenitor recruitment and vascularization in vivo. We developed an interconnective macroporous sponge from decellularized skeletal muscle with maintained biochemical traits of the intact muscle. SDF‐1α, a potent cell homing factor involved in muscle repair, was physically adsorbed or chemically immobilized in these muscle‐derived sponges. The immobilized sponges showed significantly higher SDF‐1α conjugation efficiency along with improved metabolism and infiltration of muscle‐derived stem cells in vitro, and thus generated uniform cellular constructs. In vivo, femoral muscle implantation in rats revealed a negligible immune response in all scaffold groups. We observed enhanced engraftment, neovascularization, and infiltration of CXCR4⁺ cells in the immobilized‐SDF‐1α sponge compared with nonimmobilized controls. Although Pax7⁺ cells identified adjacent to the immobilized‐SDF‐1α implantation site, other factors appear to be necessary for efficient penetration of Pax7⁺ cells into the sponge. These findings suggest that immobilization of cell homing factors via chemical mediators can result in recruitment of cells to the microenvironment with subsequent improvement in angiogenesis.