Apolipoprotein M mediates sphingosine-1-phosphate efflux from erythrocytes

Abstract Sphingosine-1-phosphate (S1P) is a bioactive lipid implicated in e.g. angiogenesis, lymphocyte trafficking, and endothelial barrier function. Erythrocytes are a main source of plasma S1P together with platelets and endothelial cells. Apolipoprotein M (apoM) in HDL carries 70% of plasma S1P, whereas 30% is carried by albumin. The current aim was to investigate the role of apoM in export of S1P from human erythrocytes. Erythrocytes exported S1P more efficiently to HDL than to albumin, particularly when apoM was present in HDL. In contrast, export of sphingosine to HDL was unaffected by the presence of apoM. The specific ability of apoM to promote export of S1P was independent of apoM being bound in HDL particles. Treatment with MK-571, an inhibitor of the ABCC1 transporter, effectively reduced export of S1P from human erythrocytes to apoM, whereas the export was unaffected by inhibitors of ABCB1 or ATPase. Thus, ABCC1 could be involved in export of S1P from erythrocytes to apoM

IL-17A potentiates TNFα-induced secretion from human endothelial cells and alters barrier functions controlling neutrophils rights of passage

Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine that regulates leukocyte mobilization and recruitment. To better understand how IL-17A controls leukocyte trafficking across capillaries in the peripheral blood circulation, we used primary human dermal microvascular endothelial cells (HDMEC) to investigate their secretory potential and barrier function when activated with IL-17A and TNFα. Activation by TNFα and IL-17A causes phosphorylation of p38 as well as IκBα whereby NFκB subsequently becomes phosphorylated, a mechanism that initiates transcription of adhesion molecules such as E-selectin. Members of the neutrophil-specific GRO-family chemokines were significantly up-regulated upon IL-17A stimulation on the mRNA and protein level, whereas all tested non-neutrophil-specific chemokines remained unchanged in comparison. Moreover, a striking synergistic effect in the induction of granulocyte colony-stimulating factors (G-CSF) was elicited when IL-17A was used in combination with TNFα, and IL-17A was able to significantly augment the levels of TNFα-induced E-selectin and ICAM-1. In accordance with this observation, IL-17A was able to markedly increase TNFα-induced neutrophil adherence to HDMEC monolayers in an in vitro adhesion assay. Using a trans-well migration assay with an HDMEC monolayer as a barrier, we here show that pre-stimulating the endothelial cells with TNFα and IL-17A together enhances the rate of neutrophil transmigration compared to TNFα or IL-17A alone. These results show that IL-17A and TNFα act in cooperation to facilitate neutrophil migration across the endothelial cell barrier. In addition, the synergistic actions of IL-17A with TNFα to secrete G-CSF appear to be important for mobilizing neutrophils from the bone marrow to the blood stream

Fibrotic Signaling in Cardiac Fibroblasts and Vascular Smooth Muscle Cells: The Dual Roles of Fibrosis in HFpEF and CAD

Patients with heart failure with preserved ejection fraction (HFpEF) and atherosclerosis-driven coronary artery disease (CAD) will have ongoing fibrotic remodeling both in the myocardium and in atherosclerotic plaques. However, the functional consequences of fibrosis differ for each location. Thus, cardiac fibrosis leads to myocardial stiffening, thereby compromising cardiac function, while fibrotic remodeling stabilizes the atherosclerotic plaque, thereby reducing the risk of plaque rupture. Although there are currently no drugs targeting cardiac fibrosis, it is a field under intense investigation, and future drugs must take these considerations into account. To explore similarities and differences of fibrotic remodeling at these two locations of the heart, we review the signaling pathways that are activated in the main extracellular matrix (ECM)-producing cells, namely human cardiac fibroblasts (CFs) and vascular smooth muscle cells (VSMCs). Although these signaling pathways are highly overlapping and context-dependent, effects on ECM remodeling mainly act through two core signaling cascades: TGF-β and Angiotensin II. We complete this by summarizing the knowledge gained from clinical trials targeting these two central fibrotic pathways

Liraglutide Reduces Both Atherosclerosis and Kidney Inflammation in Moderately Uremic LDLr-/- Mice

<div><p>Chronic kidney disease (CKD) leads to uremia. CKD is characterized by a gradual increase in kidney fibrosis and loss of kidney function, which is associated with a progressive increase in risk of atherosclerosis and cardiovascular death. To prevent progression of both kidney fibrosis and atherosclerosis in uremic settings, insight into new treatment options with effects on both parameters is warranted. The GLP-1 analogue liraglutide improves glucose homeostasis, and is approved for treatment of type 2 diabetes. Animal studies suggest that GLP-1 also dampens inflammation and atherosclerosis. Our aim was to examine effects of liraglutide on kidney fibrosis and atherosclerosis in a mouse model of moderate uremia (5/6 nephrectomy (NX)). Uremic (n = 29) and sham-operated (n = 14) atherosclerosis-prone low density lipoprotein receptor knockout mice were treated with liraglutide (1000 μg/kg, s.c. once daily) or vehicle for 13 weeks. As expected, uremia increased aortic atherosclerosis. In the remnant kidneys from NX mice, flow cytometry revealed an increase in the number of monocyte-like cells (CD68⁺F4/80^-), CD4⁺, and CD8⁺ T-cells, suggesting that moderate uremia induced kidney inflammation. Furthermore, markers of fibrosis (i.e. Col1a1 and Col3a1) were upregulated, and histological examinations showed increased glomerular diameter in NX mice. Importantly, liraglutide treatment attenuated atherosclerosis (~40%, p < 0.05) and reduced kidney inflammation in NX mice. There was no effect of liraglutide on expression of fibrosis markers and/or kidney histology. This study suggests that liraglutide has beneficial effects in a mouse model of moderate uremia by reducing atherosclerosis and attenuating kidney inflammation.</p></div

Immune cell composition is changed in kidneys from uremic LDLr-/- mice.

<p>Sixteen weeks after induction of uremia, flow cytometry was performed on kidneys from control (SHAM) and uremic (NX) LDLr-/- mice (n = 5 mice/group). The number of CD4⁺ T-cells (<b>A</b>), CD8⁺ T-cells (<b>B</b>), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (<b>C and D</b>) relative to kidney weight is depicted. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (<b>E</b>) or the M2 marker CD206 (<b>F</b>) was detected. Depicted values are mean±SEM. *p<0.05 as determined by unpaired students t-test.</p

Uremia accelerates atherosclerosis in LDLr-/- mice.

<p>Uremic (NX; n = 10) and control (SHAM; n = 10) LDLr-/- mice were put on a western type diet for 9 weeks after induction of uremia. Atherosclerosis was determined as the relative plaque area in % of the total aortic arch area. Depicted values are mean±SEM. *p<0.05 as determined by unpaired students t-test.</p

Gene expression depicted as ΔCT and fold change in NX relative to control (SHAM) in column 4 and in NX LIRA relative to NX in column 5.

<p>Results are shown as mean±SEM.</p

Uremia increases glomerular size, but not cortical collagen content in LDLr-/- mice.

<p>Representative pictures of kidney sections from control (SHAM), uremic (NX) and liraglutide treated uremic (NX LIRA) mice stained with Massons trichrome (<b>A</b>). Scale bar = 200 μm for top row and 100 μm for bottom row. Glomerular diameters were measured (<b>B</b>) (25–49 glomeruli were assessed per kidney, n = 7–8 in each group) and collagen deposition in the kidney cortex was quantified (<b>C</b>) (n = 5–7 in each group) using the Visiopharm software. **p<0.01, as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test.</p

Liraglutide attenuates NX mediated kidney inflammation in LDLr-/- mice.

<p>A-F: flow cytometry analysis of kidneys from control (SHAM; n = 7), uremic (NX; n = 7) or liraglutide treated uremic (NX LIRA; n = 7) LDLr-/- mice showing the number of CD4⁺ T-cells (<b>A</b>), CD8⁺ T-cells (<b>B</b>), monocyte-like (CD68⁺F4/80^-) and macrophage-like (CD68⁺F4/80⁺) cells (<b>C</b> and <b>D</b>) relative to kidney weight. On macrophage-like cells, median fluorescent intensity (MFI) for the M1 marker CD11c (<b>E</b>) or the M2 marker CD206 (<b>F</b>) was detected. Depicted values are mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by 1-way ANOVA followed by Sidak’s multiple comparisons post-test. n = 6 mice per group.</p