Ivo Volf

SR-BI-mediated High Density Lipoprotein (HDL) Endocytosis Leads to HDL Resecretion Facilitating Cholesterol Efflux *□

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that ∼0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.

Platelets Mediate Oxidized Low-Density Lipoprotein–Induced Monocyte Extravasation and Foam Cell Formation

Objective— A growing body of evidence indicates that platelets contribute to the onset and progression of atherosclerosis by modulating immune responses. We aimed to elucidate the effects of oxidized low-density lipoprotein (OxLDL) on platelet–monocyte interactions and the consequences of these interactions on platelet phagocytosis, chemokine release, monocyte extravasation, and foam cell formation. Approach and Results— Confocal microscopy and flow cytometric analysis revealed that in vitro and in vivo stimulation with OxLDL resulted in rapid formation of platelet–monocyte aggregates, with a preference for CD16+ monocyte subsets. This platelet–monocyte interaction facilitated OxLDL uptake by monocytes, in a process that involved platelet CD36–OxLDL interaction, release of chemokines, such as CXC motif ligand 4, direct platelet–monocyte interaction, and phagocytosis of platelets. Inhibition of cyclooxygenase with acetylsalicylic acid and antagonists of ADP receptors, P2Y1 and P2Y12, partly abrogated OxLDL-induced platelet–monocyte aggregates and platelet-mediated lipid uptake in monocytes. Platelets also enhanced OxLDL-induced monocyte transmigration across an endothelial monolayer via direct interaction with monocytes in a transwell assay. Importantly, in LDLR−/− mice, platelet depletion resulted in a significant decrease of peritoneal macrophage recruitment and foam cell formation in a thioglycollate-elicited peritonitis model. In platelet-depleted wild-type mice, transfusion of ex vivo OxLDL-stimulated platelets induced monocyte extravasation to a higher extent when compared with resting platelets. Conclusions— Our results on OxLDL-mediated platelet–monocyte aggregate formation, which promoted phenotypic changes in monocytes, monocyte extravasation and enhanced foam cell formation in vitro and in vivo, provide a novel mechanism for how platelets potentiate key steps of atherosclerotic plaque development and plaque destabilization.

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2

Summary.  Background: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram‐negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. Objectives: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. Methods: Phagocytosis, cell–cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet‐neutrophil‐bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. Results: In the presence of plasma neutrophil‐mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet–neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet–neutrophil aggregate formation but is required for efficient phagocytosis. Conclusions: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.

Urea induces macrophage proliferation by inhibition of inducible nitric oxide synthesis1

BACKGROUND Atherosclerosis is a major cause of morbidity and mortality in chronic renal failure and is associated with the proliferation of macrophages within atherosclerotic lesions. METHODS Because the progression of atherosclerosis as a consequence of decreased nitric oxide synthesis has been described, we investigated the correlation between the inhibition of inducible nitric oxide synthase (iNOS) by urea, macrophage proliferation as assayed by cell counting, tritiated thymidine incorporation and measurement of cell protein, and macrophage apoptosis. RESULTS Urea induces a dose-dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-stimulated mouse macrophages (RAW 264.7) with concomitant macrophage proliferation. Macrophage proliferation, as determined by cell counting, became statistically significant at 60 mM urea, corresponding to a blood urea nitrogen level of 180 mg/100 ml, concentrations seen in uremic patients. iNOS protein expression showed a dose-dependent reduction, as revealed by immunoblotting when cells were incubated with increasing amounts of urea. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with urea shows that the proliferative actions of urea are associated with a decrease of NO-induced apoptosis. CONCLUSIONS Our data demonstrate that the inhibition of iNOS-dependent NO production caused by urea enhances macrophage proliferation as a consequence of diminished NO-mediated apoptosis.

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the ‘classical’ scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.

Iron deficiency generates secondary thrombocytosis and platelet activation in IBD: the randomized, controlled thromboVIT trial.

Background: Secondary thrombocytosis is a common clinical feature. In patients with cancer, it is a risk factor for venous thromboembolic events. In inflammatory bowel disease (IBD), thrombocytosis is so far considered a marker of active disease and may contribute to the increased thromboembolic risk in this population. Observed effects of iron therapy on normalization of platelet counts led us to hypothesize that iron itself may regulate megakaryopoiesis. Here, we want to test the effect of iron replacement on platelet count and activity in IBD-associated thrombocytosis. Methods: We performed a randomized, single-blinded placebo-controlled trial testing the effect of ferric carboxymaltose (FCM) in patients with IBD with secondary thrombocytosis (platelets > 450 G/L). Changes in platelet counts, hemoglobin, iron parameters, disease activity, megakaryopoietic growth factors, erythropoietin, and platelet activity were assessed. Patients received placebo or up to 1500 mg iron as FCM. Endpoints were evaluated at week 6. Results: A total of 26 patients were included in the study, 15 patients were available for the per protocol analysis. A drop in platelets >25% (primary endpoint) was observed in 4 of 8 (50%, iron group) and 1 of 7 patients (14%, placebo group, P = 0.143). Mean platelet counts dropped on FCM but not on placebo (536 G/L to 411 G/L versus 580 G/L to 559 G/L; P = 0.002). Disease activity and megakaryopoietic growth factors remained unchanged and hemoglobin and iron parameters increased on FCM. The normalization of platelet counts was associated with a decrease in platelet aggregation and P-selectin expression. Conclusion: FCM lowers platelet counts and platelet activation in patients with IBD-associated secondary thrombocytosis.

Oxidation by hypochlorite converts protective HDL into a potent platelet agonist

Addition of hypochlorite‐oxidized HDL to human platelets results in an immediate and transient raise in intracellular calcium, surface expression of P‐selectin and platelet aggregation. The observed effects are dose dependent and can be blocked by an antibody directed against the lipoprotein‐binding domain of platelet thrombospondin‐ and scavenger receptor CD36.

Original ArticleUrea induces macrophage proliferation by inhibition of inducible nitric oxide synthesis1

BACKGROUND Atherosclerosis is a major cause of morbidity and mortality in chronic renal failure and is associated with the proliferation of macrophages within atherosclerotic lesions. METHODS Because the progression of atherosclerosis as a consequence of decreased nitric oxide synthesis has been described, we investigated the correlation between the inhibition of inducible nitric oxide synthase (iNOS) by urea, macrophage proliferation as assayed by cell counting, tritiated thymidine incorporation and measurement of cell protein, and macrophage apoptosis. RESULTS Urea induces a dose-dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-stimulated mouse macrophages (RAW 264.7) with concomitant macrophage proliferation. Macrophage proliferation, as determined by cell counting, became statistically significant at 60 mM urea, corresponding to a blood urea nitrogen level of 180 mg/100 ml, concentrations seen in uremic patients. iNOS protein expression showed a dose-dependent reduction, as revealed by immunoblotting when cells were incubated with increasing amounts of urea. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with urea shows that the proliferative actions of urea are associated with a decrease of NO-induced apoptosis. CONCLUSIONS Our data demonstrate that the inhibition of iNOS-dependent NO production caused by urea enhances macrophage proliferation as a consequence of diminished NO-mediated apoptosis.

Specific binding of hypochlorite-oxidized HDL to platelet CD36 triggers proinflammatory and procoagulant effects

OBJECTIVE Oxidative stress and systemic inflammation negatively affect several protective functions of high density lipoproteins (HDL) and oxidative modification of HDL by the inflammation-derived oxidant hypochlorite converts HDL into a potent platelet agonist. Therefore it was the aim of this work to clarify if these platelet-activating effects result from specific binding of hypochlorite-oxidized HDL (hyp-OxHDL) to the platelet surface and to identify responsible receptors. METHODS Binding and functional studies were performed with hyp-OxHDL in absence and presence of (potential) competitors in normal and CD36-deficient human platelets. Platelet aggregation was quantified by light transmission aggregometry. Surface expression of CD62P, phosphatidylserine and CD40L was quantified by flow cytometry. RESULTS Binding studies reveal that hyp-OxHDL show specific and saturable high-affinity binding to the platelet surface. Hyp-OxHDL trigger platelet aggregation and in a dose dependent way provoke the release of significant amounts of CD40L as well as phosphatidylserine on the platelet surface. Blocking specific binding of hyp-OxHDL to the platelet surface interferes with the ability of hyp-OxHDL to stimulate human platelets. CD36-deficient human platelets show markedly reduced binding of hyp-OxHDL. Upon addition of hypochlorite-oxidized HDL, CD36-deficient platelets do not aggregate and completely fail to release CD40L or phosphatidylserine. CONCLUSIONS From these results we conclude that specific binding of hyp-OxHDL to platelet CD36 is essential for the proinflammatory and procoagulant effects of hyp-OxHDL shown within this work. The contribution of other receptors besides CD36 to specific binding of hyp-OxHDL to the platelet membrane appears to be minimal, at best.

Hypochlorite modified LDL are a stronger agonist for platelets than copper oxidized LDL

Experimental low density lipoprotein (LDL) oxidation is usually performed using trace copper, although the in vivo relevance of this method has been called into question. Such LDL augment adenosine 5′‐diphosphate (ADP) induced platelet aggregation, presumably by the action of lipid derived compounds. In striking contrast, we find that LDL oxidized to a comparable extent by hypochlorite, an in vivo occurring oxidant, reveal themselves to be potent promoters of platelet aggregation. Interestingly, hypochlorite modified LDL seem to mediate their influence on human platelets by means of the modified apolipoprotein B‐100 (apoB) moiety. Also, the finding that hypochlorite modified albumin is able to trigger platelet aggregation suggests an essential role for hypochlorite modified protein(s) in the process of platelet activation.