Roy L. Silverstein

Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.

Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice

Macrophage scavenger receptors have been implicated as key players in the pathogenesis of atherosclerosis. To assess the role of the class B scavenger receptor CD36 in atherogenesis, we crossed a CD36-null strain with the atherogenic apo E-null strain and quantified lesion development. There was a 76.5% decrease in aortic tree lesion area (Western diet) and a 45% decrease in aortic sinus lesion area (normal chow) in the CD36-apo E double-null mice when compared with controls, despite alterations in lipoprotein profiles that often correlate with increased atherogenicity. Macrophages derived from CD36-apo E double-null mice bound and internalized more than 60% less copper-oxidized LDL and LDL modified by monocyte-generated reactive nitrogen species. A similar inhibition of in vitro lipid accumulation and foam cell formation after exposure to these ligands was seen. These results support a major role for CD36 in atherosclerotic lesion development in vivo and suggest that blockade of CD36 can be protective even in more extreme proatherogenic circumstances.

CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism

CD36, identified more than a quarter of a century ago as a platelet integral membrane glycoprotein (glycoprotein IV), was until recently best known as a receptor for thrombospondin-1 (TSP-1). TSP-1 is found in ECMs and platelet α granules, and it participates in cell attachment, motility, and proliferation, as well as in modulation of protease activity, TGF-β activation, neurite outgrowth, and angiogenesis (1). Initially, this receptor-ligand pair was shown to mediate interactions between platelets and monocytes, tumor cells, and matrix. Since then, CD36 has been implicated in multiple biological processes that define it as a multiligand scavenger receptor (see ref. 2 for review). These ligands appear remarkably diverse: In addition to TSP-1, they include long-chain fatty acids, modified LDL, retinal photoreceptor outer segments, Plasmodium falciparum malaria-parasitized erythrocytes, sickle erythrocytes, anionic phospholipids, apoptotic cells, and collagens I and IV. The biology of CD36 can be broadly divided in terms of functions that it mediates with or without TSP-1, but it is probable that it acts in concert with other proteins, such as fatty acid–binding proteins, caveola-associated proteins, integrins, cytoskeletal proteins, and signaling molecules, to effect its diverse functions.

A Null Mutation in Murine CD36 Reveals an Important Role in Fatty Acid and Lipoprotein Metabolism

A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls. Uptake of3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process. The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated.

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages.

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptakein vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75–90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

Defective Uptake and Utilization of Long Chain Fatty Acids in Muscle and Adipose Tissues of CD36 Knockout Mice

The transmembrane protein CD36 has been identified in isolated cell studies as a putative transporter of long chain fatty acids. In humans, an association between CD36 deficiency and defective myocardial uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid (BMIPP) has been reported. To determine whether this association represents a causal link and to assess the physiological role of CD36, we compared tissue uptake and metabolism of two iodinated fatty acid analogs BMIPP and 15-(p-iodophenyl) pentadecanoic acid (IPPA) in CD36 null and wild type mice. We also investigated the uptake and lipid incorporation of palmitate by adipocytes isolated from both groups. Compared with wild type, uptake of BMIPP and IPPA was reduced in heart (50–80%), skeletal muscle (40–75%), and adipose tissues (60–70%) of null mice. The reduction was associated with a 50–68% decrease in label incorporation into triglycerides and in 2–3-fold accumulation of label in diglycerides. Identical results were obtained from studies of [3H]palmitate uptake in isolated adipocytes. The block in diglyceride to triglyceride conversion could not be explained by changes in specific activities of the key enzymes long chain acyl-CoA synthetase and diacylglycerol acyltransferase, which were similar in tissues from wild type and null mice. It is concluded that CD36 facilitates a large fraction of fatty acid uptake by heart, skeletal muscle, and adipose tissues and that CD36 deficiency in humans is the cause of the reported defect in myocardial BMIPP uptake. In CD36-expressing tissues, uptake regulates fatty acid esterification at the level of diacylglycerol acyltransferase by determining fatty acyl-CoA supply. The membrane transport step may represent an important control site for fatty acid metabolism in vivo.

CD36, a Scavenger Receptor Involved in Immunity, Metabolism, Angiogenesis, and Behavior

Signaling by CD36 is ligand and cell specific and is implicated in multiple human diseases. CD36 is a multifunctional cell-surface receptor present on many cell types, including platelets, mononuclear phagocytes, and muscle, fat, and gut cells. It is conserved in mammals, and there are many invertebrate orthologs. CD36 binds to several major classes of ligands, including the matrix protein thrombospondin, long-chain fatty acids, and oxidized phospholipids and lipoproteins; in different contexts, it serves to regulate angiogenesis, innate immune responses, fatty acid metabolism, and sensory responses to fatty acids. CD36 signaling is mediated by activation of specific intracellular pathways that may include kinases of the Src family and mitogen-activated protein kinases. Because of the importance of CD36 signaling in human diseases, including atherosclerosis, thrombosis, Alzheimer’s disease, obesity, diabetes, and cancer, these signaling pathways are under intense scrutiny. This Review includes two figures and 123 citations. CD36 is a membrane glycoprotein present on platelets, mononuclear phagocytes, adipocytes, hepatocytes, myocytes, and some epithelia. On microvascular endothelial cells, CD36 is a receptor for thrombospondin-1 and related proteins and functions as a negative regulator of angiogenesis. On phagocytes, through its functions as a scavenger receptor recognizing specific oxidized phospholipids and lipoproteins, CD36 participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins, thus contributing to inflammatory responses and atherothrombotic diseases. CD36 also binds long-chain fatty acids and facilitates their transport into cells, thus participating in muscle lipid utilization, adipose energy storage, and gut fat absorption and possibly contributing to the pathogenesis of metabolic disorders, such as diabetes and obesity. On sensory cells, CD36 is involved in insect pheromone signaling and rodent fatty food preference. The signaling pathways downstream of CD36 involve ligand-dependent recruitment and activation of nonreceptor tyrosine kinases, specific mitogen-activated protein kinases, and the Vav family of guanine nucleotide exchange factors; modulation of focal adhesion constituents; and generation of intracellular reactive oxygen species. CD36 in many cells is localized in specialized cholesterol-rich membrane microdomains and may also interact with other membrane receptors, such as tetraspanins and integrins. Identification of the precise CD36 signaling pathways in specific cells elicited in response to specific ligands may yield novel targets for drug development.

Isolation of the thrombospondin membrane receptor.

Thrombospondin (TSP), a 450-kD multifunctional glycoprotein with a broad tissue distribution, is secreted upon platelet stimulation, binds to the activated platelet surface, and supports platelet aggregation. We have identified and isolated an 88-kd membrane glycoprotein present in platelets, endothelial cells, monocytes, and a variety of human tumor cell lines that is the membrane binding site for TSP. Endogenous platelet TSP binding to thrombin- and ionophore-stimulated human platelets was inhibited in the presence of the monoclonal antibody OKM5. TSP binding to C32 melanoma cells and HT1080 fibrosarcoma cells was specific and also inhibitable with OKM5 Mab. Cell labeling followed by specific immunoprecipitation demonstrated biosynthesis of a single 88-kD glycoprotein. Binding of TSP to the isolated membrane protein was specific and saturable. These studies identify an 88-kD membrane glycoprotein that reacts with the monoclonal antibody, OKM5, and may function as the cellular TSP receptor.

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgGs from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.