Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P 1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P 1 ) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P 1 ) endothelium-specific knockin of S1P 1 . The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125 I-HDL by human aortic endothelial cells was increased by an S1P 1 agonist but decreased by an S1P 1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125 I-HDL transport by the S1P 1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P 1 , apoE-haploinsufficient mice with endothelium-specific knockin of S1P 1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P 1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.

Background: The low-density lipoprotein receptor (LDLR) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of datasets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in non-transfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in non-alcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and three rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared to overexpression of wild type RBM25, overexpression of the three rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of post-transcriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

The transport of low-density lipoprotein (LDL) through the endothelium is a key step in the development of atherosclerosis, but it is notorious that phenotypic differences exist between endothelial cells originating from different vascular beds. Endothelial cells forming the blood–brain barrier restrict paracellular and transcellular passage of plasma proteins. Here, we systematically compared brain versus aortic endothelial cells towards their interaction with LDL and the role of proteins known to regulate the uptake of LDL by endothelial cells. Both brain endothelial cells and aortic endothelial cells bind and internalize LDL. However, whereas aortic endothelial cells degrade very small amounts of LDL and transcytose the majority, brain endothelial cells degrade but do not transport LDL. Using RNA interference (siRNA), we found that the LDLR–clathrin pathway leads to LDL degradation in either endothelial cell type. Both loss- and gain-of-function experiments showed that ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which promote LDL uptake and transport into aortic endothelial cells, limit neither binding nor association of LDL to brain endothelial cells. Together, these results indicate distinct LDL trafficking by brain microvascular endothelial cells and aortic endothelial cells.

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Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P<0.0001). To explore the influence of SMZL BM-MSCs in B cells survival we compared the viability of B cells isolated from the PB of healthy individuals cultured in medium alone to that of such cells co-cultured with either BM-MSCs derived from patients or normal controls. 43.85±1.46% of B cells cultured alone were apoptotic, while only 20.8±2.63% and 12±0.77% of B cells co-cultured with either normal MSCs or SMZL MSCs were apoptotic (P<0.0001 and P<0.0001, respectively). Notably patient MSCs confer a survival advantage in B cell viability over their normal counterparts (P=0.0374). Finally SMZL MSCs had a more potent chemotactic activity on B cells from healthy donors, as compared to MSCs from normal controls ( P<0.05). In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.