Narendra N. Tandon

Vascular endothelial cells synthesize and secrete brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is an abundant neurotrophin in brain and peripheral nerves, where it affects neural development, survival and repair after injury. BDNF has been detected in rat and human blood, but the source of circulating BDNF is not established. BDNF messenger and peptide were detected in cultured cells and in the culture medium of human umbilical vein endothelial cells. The expression of BDNF was up‐regulated by elevation of intracellular cAMP and down‐regulated by Ca2+ ionophore, bovine brain extract and laminar fluid shear stress. These results suggest that vascular endothelial cells may contribute to circulating BDNF.

High-shear-stress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells.

Interaction between leukocyte and endothelial cells (ECs) is essential for vascular homeostasis and competent immune-inflammatory responses in vivo. Platelet-derived microparticles (PMPs) are generated by high shear stress and may appear in diseased small arteries and arterioles in various clinical settings. In this study, we used flow cytometry and confocal laser scanning microscopy to investigate the effects of high-shear-induced platelet and microparticle activation in adhesion molecules of THP-1 and ECs. We also measured the production of some cytokines and studied cytokine mRNA from THP-1 and ECs after PMP stimulation. PMP stimulation of THP-1 cells increased CD11b, CD32, and CD33 but not CD29, CD31, and CD36. PMP stimulation of ECs increased CD54 and CD63 but not CD9, CD29, and CD31. PMPs induced interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha) production by THP-1. PMPs also induced IL-8, IL-1 beta, and interleukin-6 (IL-6) production by ECs. Production was time-dependent. With RT-PCR, some cytokine mRNAs were detected in THP-1 and ECs after PMP stimulation. In relation to adhesiveness after PMP stimulation, we could clearly observe a shift in distribution not only of CD11b in THP-1 cells but also of CD54 in ECs. In addition, anti-P-selectin glycoprotein ligand-1 antibody reduced the expression of CD11b, CD32, and CD33 in THP-1 after PMP stimulation. These results suggest that high-shear-induced microparticles may contribute to the development of atherosclerosis and participate in vascular damage in inflammatory disorders.

The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR

Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin‐stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin‐stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR‐stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR‐induced increase in both plasmalemmal proteins (FAT/CD36 (+40%) + FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue‐specific sensitivity to insulin‐induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3‐L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.

Anti‐CD36 antibodies in thrombotic thrombocytopenic purpura

Summary. The membrane glycoprotein CD36 (GPIV, Mr 88000) is found on platelets, monocytes and endothelial cells of the microvasculature. In the present study, anti CD36 antibodies have been identified as occurring with high frequency in patients with thrombotic thrombocytopenic purpura. The presence of anti CD36 antibodies in 15 TTP plasma samples thought to contain them on the basis of an initial screening by protein blots was confirmed by re‐screening against a standard of purified CD36, by immunoprecipitation from 125I‐labelled control platelets and by dot blots against purified CD36. In a further 28 random samples examined, 23/27 (85%) were CD36‐positive by immunoprecipitation, 21/28 (75%) by protein blotting, and 17/28 (60%) by dot blots against purified CD36. On protein blots following SDS‐PAGE, immunoprecipitates produced from normal platelets by TTP plasma gave positive reactions with the anti CD36 monoclonal antibody 125I‐Mo91. One half of the total TTP samples examined (21/42) caused 70% release in control platelets loaded with 14C‐serotonin. Of samples causing release 70%, one‐half (8/15) failed to cause release from Naka‐negative platelets which constitutively lack CD36 showing that CD36 was the sole target for platelet activation in these TTP samples. These studies demonstrate that antibodies directed against CD36 occur frequently in TTP patients and could cause thrombotic complications and vascular damage by reacting with the parent antigen present in platelets and endothelial cells.

Comparison of the effects of cilostazol and milrinone on intracellular cAMP levels and cellular function in platelets and cardiac cells.

Cilostazol is a potent cyclic nucleotide phosphodiesterase (PDE) type 3 (PDE3) inhibitor that was recently approved by the Food and Drug Administration (FDA) for the treatment of intermittent claudication. Its efficacy is presumed to be due to its vasodilatory and platelet activation inhibitory activities. Compared with those treated with placebo, patients treated with cilostazol showed a minimal increase in cardiac adverse events. Because of its PDE3 inhibitory activity, however, the possibility that cilostazol exerts positive cardiac inotropic effects is a safety concern. Therefore we compared the effects of cilostazol with those of milrinone, a selective PDE3 inhibitor, on intracellular cyclic adenosine monophosphate (cAMP) levels in platelets, cardiac ventricular myocytes, and coronary smooth muscle cells. We also compared the corresponding functional changes in these cells. Cilostazol and milrinone both caused a concentration-dependent increase in the cAMP level in rabbit and human platelets with similar potency. Furthermore, cilostazol and milrinone were equally effective in inhibiting human platelet aggregation with a median inhibitory concentration (IC50) of 0.9 and 2 microM, respectively. In rabbit ventricular myocytes, however, cilostazol elevated cAMP levels to a significantly lesser extent (p < 0.05 vs. milrinone). By using isolated rabbit hearts with a Langendorff preparation, we showed that milrinone is a very potent cardiotonic agent; it concentration-dependently increased left ventricular developed pressure (LVDP) and contractility. Cilostazol was less effective in increasing LVDP and contractility (p < 0.05 vs. milrinone), which is consistent with the cardiac cAMP levels. The cardiac effect of OPC-13015, a metabolite of cilostazol with about sevenfold higher PDE3 inhibition, was similar to cilostazol. Whereas milrinone concentration-dependently increased cAMP in rabbit coronary smooth muscle cells, cilostazol did not have such an effect. However, both compounds increased coronary flow equally in rabbit hearts. Our results show that although cilostazol and milrinone both inhibit PDE3, cilostazol preferentially acts on vascular elements (platelets and flow). This unique profile of cilostazol is consistent with its beneficial and safe clinical outcomes in patients with intermittent claudication.

Platelet P2Y12 Blockers Confer Direct Postconditioning-Like Protection in Reperfused Rabbit Hearts

Background: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. Methods and Results: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel’s protection. Neither aspirin nor heparin was protective. Conclusions: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC’s protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

Activation of the GP IIb-IIIa Complex Induced by Platelet Adhesion to Collagen Is Mediated by Both α2β1 Integrin and GP VI

α2β1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-α2β1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-α2β1 integrin antibody. The combination of anti-α2β1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-α2β1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by α2β1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both α2β1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.

Inhibition of platelet adhesion to collagen by monoclonal anti‐CD36 antibodies

Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non‐denaturing procedure. These antibodies reacted with control platelets, but not Naka‐negative platelets which lack CD36, as measured by flow cytometry and by immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg2+‐independent conditions but had little effect in the presence of Mg2+. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800 s−1), whereas 131.5 was without effect. These are the first anti‐CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet–collagen interaction.

Synthesis, Processing, and Intracellular Transport of CD36 during Monocytic Differentiation

CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.

Cytometric Analysis of High Shear-Induced Platelet Microparticles and Effect of Cytokines on Microparticle Generation

BACKGROUND Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation. MATERIALS AND METHODS Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry. RESULTS The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation. CONCLUSIONS These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.