A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia

Fractalkine is expressed in early and advanced atherosclerotic lesions and supports monocyte recruitment via CX3CR1

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies

FKN and CX3CR1 mediate WEHI 274.1 recruitment to injured atherosclerotic carotid arteries.

<p>(<b>A</b>) WEHI 274.1 adhesion to mechanically injured atherosclerotic carotids was studied over 30 minutes in the presence of an IgG control antibody (black bars) or a function blocking anti-FKN antibody (white bars). (<b>B</b>) Representative intravital microscopic images from mouse carotid arteries at baseline (left) and 15 minutes (right) after injury of the atherosclerotic vascular wall, pretreated with either an anti-FKN antibody (upper row) or an isotype IgG control antibody (lower row). WEHI 274.1 cells were stained with DCF (green). Bars, 50 µm. n = 4–8, *p<0.05, **p<0.01. (<b>C</b>) WEHI 274.1 adhesion is dependent on FKN-CX3CR1 interactions. WEHI 274.1 were transfected with shRNA-encoding plasmids to silence CX3CR1 expression (grey and white bars) or transfected with a plasmid encoding a scrambled control shRNA (black bars). Prior to transfusion the animals were pretreated either with a rabbit IgG control antibody (grey bars) or a function blocking anti-FKN antibody (white bars). WEHI 274.1 adhesion to injured atherosclerotic carotids was analyzed over 30 minutes.</p

Double immunofluorescence analysis of FKN and CX3CR1 expressing cells.

<p>(A) Percent distribution of FKN (green) and CX3CR1 (red) single positive and double positive (red-green stripes) cells in early (I–III) and advanced (V–VI) plaque. n = 4 carotids per group. (B) Quantitative analysis of the number of FKN and CX3CR1 positive cells in human carotid plaque. The correlation between the co-incidence of both markers was calculated by linear regression analysis. Upper panel: early plaque (I–III), r = 0.44, p<0.01. Lower panel: advanced plaque (V–VI), r = 0.74, p<0.0001. (C) A representative section from an atherosclerotic lesion (V–VI) was stained for FKN (green), CX3CR1 (red), and DAPI (blue). Bars, 100 µm. Asterisks indicate the luminal side of the vessel.</p

Monocyte and WEHI 274.1 recruitment to intact and injured atherosclerotic carotid artery lesions.

<p>(<b>A</b>) Monocyte adhesion to atherosclerotic endothelium (indicated ‘before injury’) and to the injured atherosclerotic vascular wall (indicated ‘after injury’) in ApoE^−/− mice (<b>B</b>) WEHI 274.1 adhesion to inflamed atherosclerotic endothelium and to the injured atherosclerotic vascular wall in ApoE^−/− mice. Adhesion of monocytes and WEHI 274.1 was significantly increased following injury. n = 4–8, *p<0.05, **p<0.001.</p

FKN positive structures in an advanced atherosclerotic lesion.

<p>Image of an advanced atherosclerotic plaque (VI) isolated by CEA from the human carotid artery. Insets magnify FKN positive structures within the plaque (upper row). Bottom row shows consecutive stainings for smooth muscle cells (SMA), endothelium and neovessels (Factor VIII), and macrophages (CD68). Upper scale bar: 200 µm, lower scale bar: 40 µm. Asterisk indicates the luminal side of the vessel.</p

Immunohistochemistry of FKN and CX3CR1 in atherosclerotic lesions of human carotid arteries.

<p>Representative images of human atherosclerotic plaque in different stages of lesion development (I–VI). Staining was performed with antibodies against FKN, CX3CR1, the macrophage marker CD68, and α-smooth muscle actin (SMA). Bars, 200 µm. Asterisks indicate the luminal side of the vessel.</p

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis