Yiliang Chen

CD36 and Na/K-ATPase-α1 Form a Proinflammatory Signaling Loop in Kidney

Proatherogenic, hyperlipidemic states demonstrate increases in circulating ligands for scavenger receptor CD36 (eg, oxidized low-density lipoprotein [oxLDL]) and the Na/K-ATPase (eg, cardiotonic steroids). These factors increase inflammation, oxidative stress, and progression of chronic kidney disease. We hypothesized that diet-induced obesity and hyperlipidemia potentiate a CD36/Na/K-ATPase–dependent inflammatory paracrine loop between proximal tubule cells (PTCs) and their associated macrophages and thereby facilitate development of chronic inflammation and tubulointerstitial fibrosis. ApoE -/- and apoE -/- /cd36 -/- mice were fed a high-fat diet for ⩽32 weeks and examined for physiologic and histologic changes in renal function. Compared with apoE -/-, apoE -/- /cd36 -/- mice had improved creatinine clearance and blood pressure which corresponded histologically with less glomerular and tubulointerstitial macrophage accumulation, foam cell formation, oxidant stress, and interstitial fibrosis. Coimmunopreciptation and a cell surface fluorescence-based crosslinking assay showed that CD36 and Na/K-ATPase &agr;-1 colocalized in PTCs and macrophages, and this association was increased by oxLDL or the cardiotonic steroid ouabain. OxLDL and ouabain also increased activation of Src and Lyn in PTCs. Cell-free conditioned medium from PTCs treated with oxLDL or ouabain increased macrophage migration. OxLDL, ouabain, or plasma isolated from high-fat diet–fed mice stimulated reactive oxygen species production in PTCs, which was inhibited by N-acetyl-cysteine, apocynin, or Na/K-ATPase &agr;-1 knockdown. These data suggest that ligands generated in hyperlipidemic states activate CD36 and the Na/K-ATPase and potentiate an inflammatory signaling loop involving PTCs and their associated macrophages, which facilitates the development of chronic inflammation, oxidant stress, and fibrosis underlying the renal dysfunction common to proatherogenic, hyperlipidemic states.

Oxidized LDL–bound CD36 recruits an Na+/K+-ATPase–Lyn complex in macrophages that promotes atherosclerosis

Macrophages promote atherosclerosis when an ion transporter is activated by the receptor for oxidized LDL. Na+/K+-ATPase helps turn macrophages into toxic foam cells Oxidized LDL inhibits macrophage migration and promotes lipid uptake by macrophages, which become foam cells that accumulate in atherosclerotic plaques. Chen et al. showed that CD36, the receptor for oxidized LDL, activated the tyrosine kinase Lyn in macrophages through the ion transporter Na+/K+-ATPase. Macrophages that lacked an allele encoding a subunit of the Na+/K+-ATPase were defective in responding to oxidized LDL. Apoe-null mice are prone to developing atherosclerosis when placed on a high-fat diet, and atherosclerosis development was reduced in these mice when they received macrophages lacking an allele encoding the Na+/K+-ATPase subunit. One characteristic of atherosclerosis is the accumulation of lipid-laden macrophage foam cells in the arterial wall. We have previously shown that the binding of oxidized low-density lipoprotein (oxLDL) to the scavenger receptor CD36 activates the kinase Lyn, initiating a cascade that inhibits macrophage migration and is necessary for foam cell generation. We identified the plasma membrane ion transporter Na+/K+-ATPase as a key component in the macrophage oxLDL-CD36 signaling axis. Using peritoneal macrophages isolated from Atp1a1 heterozygous or Cd36-null mice, we demonstrated that CD36 recruited an Na+/K+-ATPase–Lyn complex for Lyn activation in response to oxLDL. Macrophages deficient in the α1 Na+/K+-ATPase catalytic subunit did not respond to activation of CD36, showing attenuated oxLDL uptake and foam cell formation, and oxLDL failed to inhibit migration of these macrophages. Furthermore, Apoe-null mice, which are a model of atherosclerosis, were protected from diet-induced atherosclerosis by global deletion of a single allele encoding the α1 Na+/K+-ATPase subunit or reconstitution with macrophages that lacked an allele encoding the α1 Na+/K+-ATPase subunit. These findings identify Na+/K+-ATPase as a potential target for preventing or treating atherosclerosis.

Extracellular Vesicles Activate a CD36-Dependent Signaling Pathway to Inhibit Microvascular Endothelial Cell Migration and Tube Formation.

Objective— Literature on the effect of cell-derived extracellular vesicles (EV), ⩽1 &mgr;m vesicles shed from various cell types during activation or apoptosis, on microvascular endothelial cell (MVEC) signaling is conflicting. Thrombospondin-1 and related proteins induce anti-angiogenic signals in MVEC via CD36. CD36 binds EV via phosphatidylserine exposed on their surface but the effects of this interaction on MVEC functions are not known. We hypothesized that EV would inhibit angiogenic MVEC functions via CD36. Approach and Results— EV generated in vitro from various cell types or isolated from plasma inhibited MVEC tube formation in in vitro matrigel assays and endothelial cell migration in Boyden chamber assays. Exosomes derived from the same cells did not have inhibitory activity. Inhibition of migration required endothelial cell expression of CD36. In mouse in vivo matrigel plug assays, EV inhibited cell migration into matrigel plugs in wild type but not in cd36 null animals. Annexin V, an anionic phospholipid binding protein, when incubated with EV partially reversed inhibition of migration, suggesting a phosphatidylserine-dependent effect. EV exposure induced reactive oxygen species generation in MVEC in a NADPH oxidase and Src family kinase–dependent manner, and their inhibition by apocynin and PP2, respectively, partially reversed the EV-mediated inhibition of migration. Annexin V partially reversed EV-induced reactive oxygen species generation in murine CD36 cDNA–transfected HVUEC but not in CD36-negative human umbilical vein endothelial cell. Conclusions— These studies establish a general inhibitory effect of EV on endothelial cell proangiogenic responses and identify a CD36-mediated mechanistic pathway through which EV inhibit MVEC migration and tube formation.

Regulation of angiogenesis by phospholipid lysophosphatidic acid

Lysophosphatidic acid (LPA) as a bioactive phospholipid signaling mediator is emerging as an important regulator of endothelial cell functions and angiogenesis. Many studies have shown that LPA is an active player in regulating the processes of endothelial cell migration, proliferation, and differentiation, all essential in angiogenesis. Through modulating angiogenesis associated gene expression, LPA also promotes pathological angiogenesis. Intriguingly, the angiogenic signaling mechanisms mediated by LPA have been linked to specific G-protein coupled receptors and down stream MAPK including Erk1/2, p38 and JNK, protein kinase D (PKD-1), Rho kinase (ROCK), and the NF-kappa B signaling pathways. LPA regulates angiogenic responses via a complex signaling network, and LPA signaling is integrated and transduced to the nucleus to coordinate the transcription of different angiogenic genes. Investigation of these mechanisms will provide novel and valuable insights into the understanding of endothelial cell biology and angiogenic programs. This knowledge will facilitate designs for better therapies for the ischemic cardiovascular diseases and malignant tumors.

Acrolein Impairs the Cholesterol Transport Functions of High Density Lipoproteins

High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.

Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling

Obesity increases cancer risk including breast cancer (BC). However, the direct regulatory mechanisms by which obesity promotes BC progression remain largely unknown. We show that lysophosphatidic acid/protein kinase D1 (LPA/PKD-1)-CD36 signaling is a bona fide breast cancer promoter via stimulating microvascular remodeling in chronic diet-induced obesity (DIO). We observed that the growth of an estrogen receptor (ER) positive breast cancer was markedly increased when compared to the lean control, and specifically accompanied by increased microvascular remodeling in a syngeneic BC model in female DIO mice. The tumor neovessels in DIO mice demonstrated elevated levels of alpha smooth muscle actin (α-SMA), vascular endothelial growth factor receptor 2 (VEGFR 2) and endothelial differentiation gene 2/LPA receptor1 (Edg2/LPA1), enhanced PKD-1 phosphorylation, and reduced CD36 expression. Tumor associated endothelial cells (TAECs) exposed to LPA demonstrated sustained nuclear PKD-1 phosphorylation, and elevated mRNA levels of ephrin B2, and reduced mRNA expression of CD36. TAEC proliferation also increased in response to LPA/PKD-1 signaling. These studies suggest that the LPA/PKD-1-CD36 signaling axis links DIO to malignant progression of BC via stimulation of de novo tumor arteriogenesis through arteriolar remodeling of microvasculature in the tumor microenvironment. Targeting this signaling axis could provide an additional novel therapeutic strategy.

Platelet CD36 promotes thrombosis by activating redox sensor ERK5 in hyperlipidemic conditions

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.

Cardiotonic Steroids Stimulate Macrophage Inflammatory Responses Through a Pathway Involving CD36, TLR4, and Na/K-ATPase

Objective— Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates proinflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce proinflammatory responses in primary murine and human macrophages. Approach and Results— Using both murine peritoneal macrophages and human monocyte–derived macrophages, we demonstrated that ouabain activated NF-&kgr;B (nuclear factor kappa-light-chain-enhancer of activated B cells), leading to proinflammatory cytokine (eg, MCP-1 [monocyte chemotactic protein 1], TNF-&agr; [tumor necrosis factor-&agr;], IL-1&bgr; [interleukin-1&bgr;], and IL-6) production. By applying siRNA techniques and murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS, or fully deficient in the scavenger receptor CD36 or TLR4 (Toll-like receptor) were resistant to ouabain-induced NF-&kgr;B activation, suggesting an indispensable role of these 3 receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex, including Na/K-ATPase, CD36, and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-&kgr;B activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6C+CCR2+ monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo. Conclusions— CTS activate NF-&kgr;B leading to proinflammatory cytokine production in primary macrophages through a signaling complex, including CD36, TLR4, and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases, such as atherosclerosis.

Abstract A09: Diet-induced obesity promotes breast cancer progression by LPA-signaling-mediated functional changes of mitochondria and angiogenesis

Despite insight that obesity greatly increases cancer risk including breast cancer (BC) is supported by epidemiological studies, the direct link and mechanisms by which obesity increases BC risk remain largely unknown. Nutritional obesity is associated with a prediabetic state characterized by alteration in glucose homeostasis. The increased glucose level may aid cancer cells in generating metabolic advantage. Obesity is also accompanied by an autotaxin (ATX) mediated synthesis of bioactive signaling phospholipid or lysophosphatidic acid (LPA). ATX and plasma LPA are up-regulated in diet-induced obese (DIO) mice. We have shown that LPA stimulated angiogenesis via turning off CD36 antiangiogenic switch in endothelial cells (ECs) via protein kinase PKD-1 pathway. We hypothesize that obesity-derived LPA is a key bona fide tumor promoter by modifying mitochondrial bioenergetic metabolism and stimulating BC angiogenesis in response to high glucose. Using Seahorse Bioscience Extracellular Flux Analyzer, we discovered that LPA enhanced mitochondrial respiration of human breast adenocarcinoma cell MDA-MB231 transduced with wild type PKD-1 (PKD-WT) under high glucose conditions. LPA exposure significantly changed oxygen consumption rate (OCR), ATP linked OCAR but not extracellular acidification rate (ECAR) in cells exposed to high glucose. However, the change of OCR was attenuated by a selective PKD inhibitor CID755673. Furthermore, when exposed to conditional medium from human microvascular endothelial cells (HMVECs), MDA-MB231 overexpressing PKD-WT showed lower levels of basal OCR, ATP linked OCAR and ECAR compared with control medium. Obesity-derived LPA also inhibited CD36 expression in tumor-associated ECs and promoted BC cell growth, which was partially abolished by ATX inhibitor. Intriguingly, CID755673 inhibited clonogenic survival of MDA-MB231. To determine in vivo mechanisms of obesity-derived LPA, we established a syngeneic BC model by subcutaneously implanting E0771 adenocarcinoma in female C57BL/6J mice. DIO greatly promoted BC development in mice, demonstrated by huge tumors in DIO but not in control mice. DIO mice also showed increase in LPA receptor 1 in tumor endothelium, with robust angiogenesis and extensive bleeding inside tumor. Additionally, a high level of PD-1 expression was found in CD8 T cells inside the tumor in obese mice, implicating diet-induced obesity leads to T cell dysfunction. Our data suggests that LPA is a key player in BC angiogenesis and development but it is not the only mediator responsible for obesity-induced tumor progression. LPA-stimulated tumor progression may be associated with alternation of bioenergetic function via PKD signaling under high glucose conditions. DIO-enhanced LPA signaling may contribute to EC CD36 downregulation and dysregulated mitochondrial functions, leading to proangiogenic responses in tumor microenvironment. DIO-stimulated LPA signaling could modulate switch between mitochondrial oxidative phosphorylation and aerobic glycolysis in both BC and tumor endothelium. Targeting LPA-PKD-metabolic signaling axis may provide a novel therapeutic strategy. Citation Format: Ye Yuan, Jacob D. Kohlenberg, Yiliang Chen, Steve Komas, Gang Xin, Gloria Yuan, Weiguo Cui, Shiyong Wu, Bin Ren. Diet-induced obesity promotes breast cancer progression by LPA-signaling-mediated functional changes of mitochondria and angiogenesis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A09. doi:10.1158/1538-7445.CHTME14-A09

Platelet metabolism meets thrombosis

Metabolic pathways intersect with many processes important in hematology, including thrombosis, innate and adaptive immunity, malignant transformation, and stem cell function. These pathways are increasingly recognized as potentially targetable for therapeutic intervention. In this issue of Blood , Lepropre et al identify the energy sensor AMP-activated protein kinase (AMPK) in platelets as a regulator of thrombosis. 1