Martin G. Sirois

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

Background— Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results— Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions— Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.

AIMSnCD36 has been shown to associate with non-receptor Src kinases to activate mitogen-activated protein kinases and trigger cytoskeletal remodelling, important events in foam cell formation and macrophage migration. Yet, its role in regulating circulating mononuclear phagocyte trafficking to atherosclerotic lesions has not been investigated. The aim of the present study was to investigate the role of CD36 in modulating the recruitment of mononuclear phagocytes to the arterial wall and the associated vascular inflammation, using both pharmacological and genetic approaches.nnnMETHODS AND RESULTSnApolipoprotein E-deficient (apoE(-/-)) mice fed a high-fat, high-cholesterol diet were treated daily with a CD36 ligand, EP 80317 (300 microg/kg), or 0.9% NaCl for 6 or 12 weeks. Forty-eight hours before sacrifice, mice were injected iv with (111)Indium-labelled macrophages. A 65% (P < 0.001) reduction of labelled macrophage accumulation at aortic lesions was observed in EP 80317-treated mice, mainly at the level of the aortic arch and iliac arteries, correlating with a 43% reduction of atherosclerotic lesion areas. This was associated with reduced phosphorylation of the focal adhesion kinase Pyk2 following stimulation with oxidized phospholipid in a Src kinase- and CD36-dependent manner. At the vascular level, EP 80317 treatment reduced the expression of pro-inflammatory proteins, including NADPH oxidase, inducible nitric oxide synthase, vascular endothelial cell adhesion molecule-1, and CCL2 chemokine. Plasma IL-6 levels were also reduced by 40% (P < 0.05). In contrast, none of these proteins was modulated in EP 80317-treated apoE/CD36 double knockout (apoE(-/-)/CD36(-/-)) mice.nnnCONCLUSIONnOur results support a role for CD36 signalling in the regulation of mononuclear phagocyte trafficking to atherosclerotic-prone sites and in the associated vascular wall inflammation.

HIF2α reduces growth rate but promotes angiogenesis in a mouse model of neuroblastoma

BackgroundHIF2α/EPAS1 is a hypoxia-inducible transcription factor involved in catecholamine homeostasis, vascular remodelling, physiological angiogenesis and adipogenesis. It is overexpressed in many cancerous tissues, but its exact role in tumour progression remains to be clarified.MethodsIn order to better establish its function in tumourigenesis and tumour angiogenesis, we have stably transfected mouse neuroblastoma N1E-115 cells with the native form of HIF2α or with its dominant negative mutant, HIF2α (1–485) and studied their phenotype in vitro and in vivo.ResultsIn vitro studies reveal that HIF2α induces neuroblastoma cells hypertrophy and decreases their proliferation rate, while its inactivation by the HIF2α (1–485) mutant leads to a reduced cell size, associated with an accelerated proliferation. However, our in vivo experiments show that subcutaneous injection of cells overexpressing HIF2α into syngenic mice, leads to the formation of tumour nodules that grow slower than controls, but that are well structured and highly vascularized. In contrast, HIF2α (1–485)-expressing neuroblastomas grow fast, but are poorly vascularized and quickly tend to extended necrosis.ConclusionTogether, our data reveal an unexpected combination between an antiproliferative and a pro-angiogenic function of HIF2α that actually seems to be favourable to the establishment of neuroblastomas in vivo.

Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells

Plasmacytoid dendritic cells (pDCs) are unique bone-marrow-derived cells that produce large amounts of type I interferon in response to microbial stimulation. Furthermore, pDCs also promote Txa0cell tolerance in sterile-inflammation conditions. However, the immunomodulatory role of aortic pDCs in atherosclerosis has been poorly understood. Here, we identified functional mouse and human pDCs in the aortic intima and showed that selective, inducible pDC depletion in mice exacerbates atherosclerosis. Aortic pDCs expressed CCR9 and indoleamine 2,3-dioxygenase 1 (IDO-1), an enzyme involved in driving the generation of regulatory Txa0cells (Tregs). As a consequence, loss of pDCs resulted in decreased numbers of Tregs and reduced IL-10 levels in the aorta. Moreover, antigen presentation by pDCs expanded antigen-specific Tregs in the atherosclerotic aorta. Notably, Tregs ablation affected pDC homeostasis in diseased aorta. Accordingly, pDCs in human atherosclerotic aortas colocalized with Tregs. Collectively, we identified a mechanism of atheroprotection mediated by tolerogenic aortic pDCs.

Group V secreted phospholipase A2 contributes to LPS‐induced leukocyte recruitment

Secreted phospholipases A2 (sPLA2s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA2 isoforms are up‐regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA2 (sPLA2‐V). Furthermore, it has recently been shown that sPLA2‐V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA2‐V on LPS‐mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA2‐V null mice (sPLA2‐V−/−) and control wild‐type (WT) littermates. We observed that LPS (1u2009µg/ml)‐mediated leukocyte emigration in sPLA2‐V−/− was attenuated by 52% and 86% upon 6 and 12u2009h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell‐permeable sPLA2 inhibitor (12‐epi‐scalaradial; SLD) reduced LPS‐mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA2‐V−/− mice. Protein analyses from the air pouch skin were carried out upon LPS‐challenge, and the expression of intercellular adhesion molecule (ICAM)‐1 and vascular cell adhesion molecule (VCAM)‐1 were both significantly reduced in sPLA2‐V−/− mice as compared to control WT mice. Together, our data demonstrate the role of sPLA2‐V in LPS‐induced ICAM‐1 and VCAM‐1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA2‐V in the development of inflammatory innate immune responses. J. Cell. Physiol. 224:127–134, 2010

Angiopoietin-1 but not angiopoietin-2 induces IL-8 synthesis and release by human neutrophils.

We previously reported Tie2 receptor expression on human neutrophils, which promote chemotactic activities upon activation by both angiopoietins (Ang1 and Ang2). Moreover, we observed that neutrophil pretreatment with Ang1 or Ang2 enhances interleukin‐8 (IL‐8) chemotactic effect. Therefore, we assessed the capacity of Ang1 and/or Ang2 to modulate neutrophil IL‐8 synthesis and release. Neutrophils isolated from healthy donors were stimulated in a time‐ (1–6u2009h) and concentration‐(10−10–10−8u2009M) dependent manner with both angiopoietins. IL‐8 mRNA production was measured by RT‐qPCR, whereas its protein synthesis and release from neutrophils was assessed by ELISA. Ang1 (10−8u2009M) induced a significant and maximal increase of IL‐8 mRNA (4.7‐fold) within 1u2009h, and promoted maximal IL‐8 protein synthesis (3.6‐fold) and release (5.5‐fold) within 2u2009h as compared to control PBS‐treated neutrophils. Treatment with Ang2 alone did not modulate IL‐8 synthesis or release, and its combination to Ang1 did not affect Ang1 activity. Neutrophil pretreatment with a protein synthesis inhibitor (CHX) increased IL‐8 mRNA synthesis by 18‐fold, and reduced Ang1‐mediated IL‐8 protein synthesis and release by 96% and 92%, respectively. Pretreatment with a transcription inhibitor (ActD) reduced IL‐8 mRNA synthesis by 54% and IL‐8 protein synthesis and release by 52% and 79%, respectively. Using specific kinase inhibitors, we observed that Ang1‐driven IL‐8 mRNA and protein synthesis is p42/44 MAPK‐dependent and ‐independent from p38 MAPK and PI3K activity. Our study is the first to report the capacity of Ang1 (as opposed to Ang2) to promote neutrophil IL‐8 synthesis and release through the activation of p42/44 MAPK pathway. J. Cell. Physiol. 227: 3099–3110, 2012.

VEGF and Angiopoietins Promote Inflammatory Cell Recruitment and Mature Blood Vessel Formation in Murine Sponge/Matrigel Model

A key feature in the induction of pathological angiogenesis is that inflammation precedes and accompanies the formation of neovessels as evidenced by increased vascular permeability and the recruitment of inflammatory cells. Previously, we and other groups have shown that selected growth factors, namely vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) do not only promote angiogenesis, but can also induce inflammatory response. Herein, given a pro‐inflammatory environment, we addressed the individual capacity of VEGF and angiopoietins to promote the formation of mature neovessels and to identify the different types of inflammatory cells accompanying the angiogenic process over time. Sterilized polyvinyl alcohol (PVA) sponges soaked in growth factor‐depleted Matrigel mixed with PBS, VEGF, Ang1, or Ang2 (200u2009ng/200u2009µl) were subcutaneously inserted into anesthetized mice. Sponges were removed at day 4, 7, 14, or 21 post‐procedure for histological, immunohistological (IHC), and flow cytometry analyses. As compared to PBS‐treated sponges, the three growth factors promoted the recruitment of inflammatory cells, mainly neutrophils and macrophages, and to a lesser extent, T‐ and B‐cells. In addition, they were more potent and more rapid in the recruitment of endothelial cells (ECs) and in the formation and maturation (ensheating of smooth muscle cells around ECs) of neovessels. Thus, the autocrine/paracrine interaction among the different inflammatory cells in combination with VEGF, Ang1, or Ang2 provides a suitable microenvironment for the formation and maturation of blood vessels. J. Cell. Biochem. 116: 45–57, 2015.

Role of MSK1 in the signaling pathway leading to VEGF-mediated PAF synthesis in endothelial cells.

Vascular endothelial growth factor (VEGF) inflammatory effects require acute platelet‐activating factor (PAF) synthesis by endothelial cells (EC). We previously reported that VEGF‐mediated PAF synthesis involves the activation of VEGF receptor‐2/Neuropilin‐1 complex, which is leading to the activation of p38 and p42/44 mitogen‐activated protein kinases (MAPKs) and group V secretory phospholipase A2 (sPLA2‐V). As the mechanisms regulating sPLA2‐V remain unknown, we addressed the role of the mitogen‐ and stress‐activated protein kinase‐1 (MSK1), which can be rapidly and transiently activated by p38 or p42/44 MAPKs. In native bovine aortic endothelial cells (BAEC), we observed a constitutive protein interaction of MSK1 with p38, p42/44 MAPKs, and sPLA2‐V. These protein interactions were maintained in BAEC transfected either with the empty vector pCDNA3.1, wild‐type MSK1 (MSK1‐WT) or N‐terminal dead kinase MSK1 mutant (MSK1‐D195A). However, in BAEC expressing C‐terminal dead kinase MSK1 mutant (MSK1‐D565A), the interaction between MSK1 and sPLA2‐V was reduced by 82% and 90% under basal and VEGF‐treated conditions as compared to native BAEC. Treatment with VEGF for 15 min increased basal PAF synthesis in native BAEC, pCDNA3.1, MSK1‐WT, and MSK1‐D195A by 166%, 139%, 125%, and 82%, respectively. In contrast, PAF synthesis was prevented in cells expressing MSK1‐D565A mutant. These results demonstrate the essential role of the C‐terminal domain of MSK1 for its constitutive interaction with sPLA2‐V, which appears essential to support VEGF‐mediated PAF synthesis. J. Cell. Biochem. 98: 1095–1105, 2006.

Angiopoietin-1 Upregulates De Novo Expression of Il-1β and Il1-Ra, and the Exclusive Release of Il1-Ra from Human Neutrophils

The expression of the angiopoietin (Ang) receptor, Tie2, on both endothelial and inflammatory cells supports the idea that Ang signaling may play a fundamental role in initiating and maintaining the inflammatory response. We have previously shown that Ang1 and/or Ang2 alter the innate immune response by enhancing human neutrophil survival, chemotaxis and production of inflammatory cytokine interleukin-8 (IL-8) in vitro. Thus, we hypothesized that Ang1 and Ang2 could modulate other inflammatory signals in neutrophils, a possibility we explored through a gene-based assay looking at changes in the mRNA expression of 84 inflammatory cytokines and their receptors. We observed that Ang1 (10−8 M), but not Ang2, increased mRNA expression of prominent pro-inflammatory cytokine IL-1β and its natural antagonist IL-1RA, by up to 32.6- and 10.0-fold respectively, compared to PBS-control. The effects of Ang1 extended to the proteins, as Ang1 increased intracellular levels of precursor and mature IL-1β, and extracellular levels of IL-1RA proteins, by up to 4.2-, 5.0- and 4.4-fold respectively, compared to PBS-control. Interestingly, Ang1 failed at inducing IL-1β protein release or at increasing intracellular IL-1RA, but the ratio of IL-1RA to mature IL-1β remained above 100-fold molar excess inside and outside the cells. The above-noted effects of Ang1 were mediated by MAP kinases, whereby inhibiting MEK1/2 lead to up to 70% effect reduction, whereas the blockade of p38MAPK activity doubled Ang1s effect. These findings suggest that Ang1 selectively alters the balance of neutrophil-derived inflammatory cytokines, favoring the blockade of IL-1 activity, a consideration for future therapies of inflammatory diseases.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1β Synthesis and Release.

We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2) to induce pro-inflammatory activities, such as platelet-activating factor synthesis, β2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1β (MIP-1β) by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1β protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1β protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM) increased neutrophil intracellular and extracellular MIP-1β concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002), p38 MAPK (SB203580) and MEK (U0126) inhibitors completely inhibited Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide), did also abrogate Ang1-mediated increase of MIP-1β intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1β mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1β gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.