Higher CD36 Expression Research Articles

CD36 promotes vasculogenic mimicry in melanoma by mediating adhesion to the extracellular matrix

BackgroundThe formation of blood vessels within solid tumors directly contributes to cancer growth and metastasis. Until recently, tumor vasculature was thought to occur exclusively via endothelial cell (EC) lined structures (i.e. angiogenesis), but a second source of tumor vasculature arises from the cancer cells themselves, a process known as vasculogenic mimicry (VM). While it is generally understood that the function of VM vessels is the same as that of EC-lined vessels (i.e. to supply oxygen and nutrients to the proliferating cancer cells), the molecular mechanisms underpinning VM are yet to be fully elucidated.MethodsHuman VM-competent melanoma cell lines were examined for their VM potential using the in vitro angiogenesis assays (Matrigel), together with inhibition studies using small interfering RNA and blocking monoclonal antibodies. Invasion assays and adhesion assays were used to examine cancer cell function.ResultsHerein we demonstrate that CD36, a cell surface glycoprotein known to promote angiogenesis by ECs, also supports VM formation by human melanoma cancer cells. In silico analysis of CD36 expression within the melanoma cohort of The Cancer Genome Atlas suggests that melanoma patients with high expression of CD36 have a poorer clinical outcome. Using in vitro ‘angiogenesis’ assays and CD36-knockdown approaches, we reveal that CD36 supports VM formation by human melanoma cells as well as adhesion to, and invasion through, a cancer derived extracellular matrix substrate. Interestingly, thrombospondin-1 (TSP-1), a ligand for CD36 on ECs that inhibits angiogenesis, has no effect on VM formation. Further investigation revealed a role for laminin, but not collagen or fibronectin, as ligands for CD36 expressing melanoma cells.ConclusionsTaken together, this study suggests that CD36 is a novel regulator of VM by melanoma cancer cells that is facilitated, at least in part, via integrin-α3 and laminin. Unlike angiogenesis, VM is not perturbed by the presence of TSP-1, thus providing new information on differences between these two processes of tumor vascularization which may be exploited to combat cancer progression.

Prognostic and immunological role of CD36: A pan-cancer analysis.

CD36 plays a critical role in lipid metabolism, which is closely associated with human immunity. However, the role of CD36 in cancer remains unclear. We performed a pan-cancer analysis to elucidate the potential role of CD36 in cancer by investigating its prognostic value and current predictors for the efficacy of immune checkpoint inhibitors (ICIs) in multiple cancer types. CD36 expression in cancer cell lines, tumor tissue, and their adjacent normal tissues displayed heterogeneity among different cancers. Immunohistochemistry was used to detect CD36 expression and confirmed the results. CD36 expression significantly affects prognosis in the six cancer types. High CD36 expression was marginally associated with poorer prognosis in four of them and improved prognosis in the remaining two types. CD36 expression was significantly correlated with the 6 immune infiltrates in most cancer types. In addition, CD36 gene expression was positively correlated with Stromal score, Immune score, and ESTIMATE score. A total of 47 immune checkpoint genes were collected and their relationship with CD36 expression was analyzed. CD36 expression was significantly associated with multiple stimulatory and inhibitory checkpoint molecules with a disease-specific pattern. As to the genes reported to positively relate to the efficacy of ICIs, CD36 expression was positively correlated with most of them but negatively associated with a small proportion of cancer type-specific patterns. Concerning the genes negatively related to the efficacy of ICIs, CD36 expression was positively correlated with NRP1 and TNFSF15 in multiple cancers. CD36 expression was negatively correlated with tumor neoantigen burden in most cancer types. However, CD36 expression was negatively correlated with tumor mutation burden in most cancer types. The correlation between CD36 expression and the four methyltransferases was also significant in multiple cancers, but also with a cancer type-specific pattern. In summary, the current study found CD36 expression and its prognostic value in multiple cancer types. In addition, the expression of CD36 was significantly associated with current predictors for the efficacy of ICIs. The practical application value of CD36 is disease specific.

CD36 upregulates DEK transcription and promotes cell migration and invasion via GSK-3β/β-catenin-mediated epithelial-to-mesenchymal transition in gastric cancer.

Evidence indicates that the lipid scavenger receptor CD36 has pro-metastatic functions in several cancers. Although CD36 expression correlates with an unfavorable prognosis in gastric cancer (GC), its specific contribution to disease onset, progression, and/or metastasis remains unclear. Using bioinformatics analyses, we ascertained that CD36 expression was increased in metastatic GC specimens in The Cancer Genome Atlas and Gene Expression Omnibus databases and correlated with poor prognosis. In addition, higher CD36 expression was associated with lymph node metastasis (p < 0.05) and poor prognosis (p = 0.030) in 79 Chinese GC patients. Basal CD36 expression levels correlated positively with migration, invasion, and expression of epithelial-to-mesenchymal transition (EMT) markers in GC cell lines, a relationship confirmed by knockdown and overexpression experiments. Importantly, analysis of gene expression changes in CD36-knockdown GC cells led us to identify the chromatin-associated protein DEK as a c-Myc target that mediates activation of the GSK-3β/β-catenin signaling pathway to trigger EMT. These findings further our understanding of the mechanisms governing metastatic dissemination of GC cells and suggest the therapeutic potential of strategies targeting CD36.

CD36 Expression Is Associated with Cancer Aggressiveness and Energy Source in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an important cause of cancer-related death worldwide. CD36, a long-chain fatty acid (FA) receptor, can initiate metastasis in human oral squamous cell carcinoma (SCC), and its expression is associated with poor prognosis in several cancers. The clinical significance of CD36 expression and its function in ESCC remain unknown. We examined the clinical significance of CD36 expression in 160 ESCC samples using immunohistochemical staining. Functional analysis was performed to determine the association between CD36 and ESCC characteristics (proliferative ability, invasive ability, and energy source dependency). Thirty (18.8%) ESCC cases showed high CD36 expression, indicating a significant association with progression. CD36 suppression inhibited proliferation and invasiveness in ESCC cells. ESCC cells with CD36 suppression used specific essential amino acids (EAAs) as energy sources. Cell viability depended on FAs under CD36 expression. The viability of ESCC cells with CD36 suppression depended on EAAs but not FAs. CD36 may be a good biomarker and therapeutic target in ESCC. Our data provide new insights into the basic mechanism of CD36-dependent energy utilization for ESCC survival. CD36 might be a key regulator of the dependency of FAs as energy source in ESCC cells.

The role of CD36 in macrophage lipid metabolism and function in tumor microenvironment

Abstract Macrophages are a highly plastic population of innate immunity with diverse functions in homeostasis. Despite their relative abundance in tumor microenvironment (TME), the exact function of tumor-associated macrophages (TAMs) and how they are regulated remain largely unknown. Macrophages also play key roles in lipid metabolism, disruption of which can result in pathologies such as atherosclerosis. Interestingly, TME has also been characterized as a lipid-rich context, we are interested in understanding how lipids in TME affect TAMs’ pro- or anti-tumor function. Here, we firstly characterized the lipid metabolic phenotype of TAMs in a mouse melanoma model. We found that up-regulation of lipid storage and uptake is associated with the most tolerogenic TAM subset marked by high level of F4/80. We also found high expression of scavenger receptor CD36 on F4/80hi TAMs which mediates uptake of oxidized LDL (oxLDL) from TME. Using germ line knockout (Cd36−/−) and myeloid-specific deficiency (Cd36 flox/flox x Csf1r-Cre) mouse models, we observed that uptake of oxLDL by TAMs was significantly blocked, along with rescued immunosuppressive phenotype characterized by decreased expression of PD-L1, CD206 and elevated secretion of TNFa, which together contribute to slower tumor growth. We further conducted single-cell RNA sequencing on Cd36−/− and wild-type TAMs, and found several pathways as potential mechanisms for CD36-mediated pro-tumor phenotype. In conclusion, we discovered that high expression of CD36 on TAMs mediates uptake of oxLDL and contribute to pro-tumor function. Our data suggests that metabolites in TME may play crucial roles in metabolic and functional reprogramming of tumor-infiltrating immune populations.

Lipid accumulation impairs natural killer cell cytotoxicity and tumor control in the postoperative period

BackgroundNatural killer (NK) cell dysfunction following cancer surgery has been shown to promote metastases. Recent studies demonstrate an emerging role for lipids in the modulation of NK cell innate responses. However, the mechanisms involved in lipid modulation of NK cell postoperative anti-tumor function are unknown. This current study will determine whether the lipid accumulation via scavenger receptors on NK cells is responsible for the increase in postoperative metastasis.MethodsLipid content in mouse and human NK cells was evaluated by flow cytometry. NK cell scavenger receptor (SR) expression was measured by microarray analysis, validated by qRT-PCR and flow cytometry. NK cell ex vivo and in vivo tumor killing was measured by chromium-release and adoptive transfer assays, respectively. The mediating role of surgery-expanded granulocytic myeloid derived suppressor cells (gMDSC) in SR induction on NK cells was evaluated using co-culture assays.ResultsNK cells in surgery-treated mice demonstrated increased lipid accumulation, which occurred via up-regulation of MSR1, CD36 and CD68. NK cells with high lipid content had diminished ability to lyse tumor targets ex vivo. Adoptive transfer of lipid-laden NK cells into NK cell-deficient mice were unable to protect against a lung tumor challenge. Granulocytic MDSC from surgery-treated mice increased SR expression on NK cells. Colorectal cancer surgical patients showed increased NK cell lipid content, higher CD36 expression, decreased granzyme B and perforin production in addition to reduced cytotoxicity in the postoperative period.ConclusionsPostoperative lipid accumulation promotes the formation of metastases by impairing NK cell function in both preclinical surgical models and human surgical colorectal cancer patient samples. Understanding and targeting the mechanisms underlying lipid accumulation in innate immune NK cells can improve prognosis in cancer surgical patients.

Abstract A223: CD36-mediated metabolic adaptation guides regulatory T-cells in tumors

Abstract Regulatory T-cells (Tregs) play an indispensable role in maintaining peripheral tolerance and preventing autoimmune disease. In addition to modulating tissue homeostasis, the suppressive properties of Tregs can also be harnessed by cancers to evade immunosurveillance. Therefore, depleting Tregs has been shown to unleash antitumor immunity and interrupt formation of an immunosuppressive tumor microenvironment (TME). However, systemic loss of Tregs due to Treg depletion also leads to severe autoimmunity. Therefore, the identification of novel approaches that specifically target intratumoral Tregs is direly needed for unleashing antitumor immunity and cancer immunotherapy. Here we show that intratumoral Tregs increase lipid uptake and content and elevated expression of CD36, a fatty acid translocase, as compared to Tregs in circulation and other normal tissues, in several cancer types. By using the transgenic mice model, we found that Treg-specific ablation of CD36 reduces accumulation of intratumoral Treg and suppresses tumor growth. Importantly, Treg-specific CD36 deficiency does not lead to autoimmunity in aged mice and CD36-deficient Tregs remain their suppressive activity on restraining CD4 T-cell-induced inflammatory bowl disease. Mechanistically, CD36 expression supports survival of intratumoral Tregs by fine-tuning their mitochondrial fitness via PPAR signaling. Thus, high expression of CD36 in intratumoral Tregs orchestrates Treg metabolic adaptation in tumors by intervening metabolic regulations, and further promotes tumor growth by suppressing the antitumor immune responses. Ultimately, anti-PD-1 blockade treatment elicits therapeutic benefits in mice with Treg-specific ablation of CD36. Altogether, our study suggests that CD36 might be a potential target for specifically waning down intratumoral Tregs and provide proof-of-concept evidence that targeting CD36 in tumors could unleash antitumor immunity and synergize with checkpoint blockade treatment. Citation Format: Haiping Wang, Ping-Chih Ho. CD36-mediated metabolic adaptation guides regulatory T-cells in tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A223.

Fatty acid-induced CD36 expression via O-GlcNAcylation drives gastric cancer metastasis.

Metastasis is the primary cause of death in patients with advanced cancer. Recently, a high-fat diet was shown to specifically promote the metastatic potential of specific cancer cells in a CD36-dependent manner. However, the molecular basis of the fatty acid (FA)-induced upregulation of CD36 has remained unclear.Methods: RT-qPCR, FACS analysis, immunoblotting and immunohistochemistry, as well as retrieving TCGA database, were carried out to quantitate CD36 expression in gastric cancer (GC) tissues and cell lines. Transwell assay and xenografts were used to assess cell metastasis abilities in vitro and in vivo after indicated treatment. Luciferase reporter assay was carried out to evaluate the changes in signaling pathways when O-GlcNAcylation level was increased in GC cells and in vitro O-GlcNAcylation assay was utilized for wild and mutant types of CD36 protein to explore the potential O-GlcNAcylation sites.Results: High CD36 expression is a predictor of poor survival and promotes metastasis of GC cells and the use of neutralizing antibodies to block CD36 inhibits GC metastasis in mice. FA or a HFD promotes the metastatic potential of GC cells by upregulating CD36 via increasing the O-GlcNAcylation level. Increased O-GlcNAcylation levels promote the transcription of CD36 by activating the NF-κB pathway and also increase its FA uptake activity by directly modifying CD36 at S468 and T470.Conclusion: FA-induced hyper-O-GlcNAcylation promotes the transcription and function of CD36 by activating the NF-κB pathway and directly modifying CD36 at S468 and T470, which drives GC metastasis.

Chronic Lymphocytic Leukaemia Relies on Lipid Scavenging and Synthesis As an Energy Source

Dysregulation of cancer cell bioenergetics is one of the hallmarks of cancer. The Warburg effect is one such documented change. However, glucose metabolism is not universally increased in cancer cells. Uptake of radiolabelled glucose in chronic lymphocytic leukaemia (CLL) fails as a marker of proliferation and the underlying reason is not well elucidated [Conte et al, 2014]. Using proteomic and comprehensive lipid analyses, the preferred metabolic pathways of CLL cells have been identified.We have previously shown by proteomic analysis that circulating peripheral blood CLL cells demonstrate a significant increase in the expression of proteins pivotal in endogenous lipid synthesis pathways compared to B-cells from healthy individuals. These include fatty acid synthase (+3.84 fold), farnesyl diphosphate synthase (+2.17 fold), ATP-citrate lyase (+4.63 fold), citrate synthase (+2.45 fold) and acetyl-CoA acetyltransferase 1 (+5.84 fold). Conversely, analysis of the proliferation centres in CLL lymph nodes reveals increased expression of proteins involved in beta-oxidation, such as hydroxyacyl-CoA dehydrogenase (+2.02 fold). Additionally CLL cells shows a large increase in the levels of phospholipids found associated with lipid droplets compared to healthy B-cells, including phosphatidylinositol and phosphatidylethanolamine.Our current studies have expanded these observations in an attempt to determine if CLL cells have a preponderance for endogenous synthesis of lipids or exogenous uptake. We performed a comprehensive analysis of CLL cells from the bone marrow and peripheral blood using a variety of techniques. Transmission electron microscopy (TEM) images demonstrate striking morphological differences between normal B-cells and CLL cells with the inclusion of lipid droplets in the cytoplasm of CLL cells (Figure 1A) which was most evident in peripheral CLL samples. These were confirmed to be lipid droplets by the specific marker Bodipy 493/503 on flow cytometry (Fig. 1B) and confocal microscopy imaging with ReZolveL1 which targets neutral lipids (Fig. 1C). These showed a substantially higher level of lipid droplet staining in CLL cells compared to normal B-cells. Additionally there was higher expression of CD36 in CLL cells which is the receptor for exogenous lipid uptake into cells (Fig 1D).TEM analysis also revealed a high number of lysosomes in CLL cells, which was confirmed with lysotracker imaging on confocal microscopy (Fig. 1E). Lysosomes are now emerging as key players in lipid transport and biogenesis [Thelen and Zoncu, 2017]. Lipid droplets may be broken down by a process called autophagy mediated lipid degradation, or lipophagy, which relies on autophagosomes delivering lipid droplets to lysosomes. Autophagosomes were observed in several further bone marrow samples (Fig. 1F). The bone marrow samples also showed evidence of ‘condensed mitochondria’, indicative of high rates of beta-oxidation [Rossignol et al, 2004].qPCR was then carried out on 90 genes involved in lipid metabolism. These results revealed significant differences between healthy B-cells and CLL cells. All CLL samples analysed demonstrated high expression of PLIN1, which protects lipid droplets from degradation, and low expression of APOC1, which prevents fatty acid uptake. From these observations we propose that peripheral CLL cells scavenge lipids from the periphery, mainly by uptake via the CD36 receptor, and increased endogenous lipid synthesis pathways.These results underpin a model for metabolism and enhanced survival for CLL cells in the hypoxic and potentially nutrient poor bone marrow/lymph node microenvironments. Peripheral CLL cells scavenge lipids; these excess lipids, which are toxic to the cell, are stored in lipid droplets which are protected from degradation by a high expression of PLIN proteins. Once these cells circulate back to the bone marrow and lymph nodes to proliferate, the lipid droplets are degraded, likely by lipophagy and neutral lipolysis which frees fatty acids to be used in beta oxidation in the mitochondria to sustain cell proliferation in the bone marrow and lymph node compartments. Our results begin to unravel CLL bioenergetics and dysregulation of cellular metabolism that occurs in this disease. We are now investigating whether the manipulation of these pathways, particularly lipophagy, may represent a novel therapeutic approach in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Can expression of CD36 be a metabolic marker of survival in muscle invasive urothelial carcinoma of the bladder?

Background: There is continued interest in cancer metabolism due to its potential application in targeted cancer therapies. One of the promising lines of research is concerned with the CD36 protein which is involved in numerous metabolic processes. The role of CD36 in the regulation of angiogenesis and transportation of fatty acids into the cell was demonstrated, both of which are implicated in tumor development. However, the prognostic value of CD36 in urinary bladder cancer (UBC) has not been previously evaluated. The aim of this study was to assess the relation between CD36 expression in tumor cells and survival of patients with pT2-pT4 UBC. Materials and methods: 96 patients with stage pT2-pT4 UBC who underwent radical cystectomy/cystoprostatectomy between 2011 and 2014 were included in the study. Archival paraffin blocks which were the most representative of tumor were selected based on H&E stained samples. Immunostaining was performed with a recombinant monoclonal rabbit anti-CD36 antibody (EPR6573, abcam). In each sample the intensity of CD36 expression was assessed on a binary scale as either high or low. Moreover, the extent of CD36 expression within the tumors was assessed as low in tumors with fewer than 30% of TCs expressing CD36 and high in tumors with more than 30% of the tumor cells expressing CD36. CD36 expression and the probability of survival were analyzed with Kaplan–Meier curves. Results: We observed an over two-fold decrease in 5-year survival of patients with high expression of CD36 compared to patients with low expression (p=0.004). Similarly, 2-year survival of patients with more than 30% of the tumor cells expressing CD36 was almost two-fold lower compared to patients with the extent of CD36 expression below 30% (p=0.028). Conclusions: The expression of CD36 in tumor cells allows for the stratification of survival in patients with UBC. The intensity and extent of CD36 expression may be considered as new prognostic factors which describe the metabolic status of cancer cells and their resulting malignant potential. Further research leading to the development of a model of metabolic tumor progression that incorporates CD36 expression could open new therapeutic opportunities for UBC patients.

Simvastatin Promotes Hematoma Absorption and Reduces Hydrocephalus Following Intraventricular Hemorrhage in Part by Upregulating CD36.

We previously found that hematoma worsens hydrocephalus after intraventricular hemorrhage (IVH) via increasing iron deposition and aggravating ependymal cilia injury; therefore, promoting hematoma absorption may be a promising strategy for IVH. Recently, some investigations imply that simvastatin has the ability of accelerating hematoma absorption. Thus, this study was designed to examine the efficacy of simvastatin for IVH in rats. Intracerebral hemorrhage with ventricular extension was induced in adult male Sprague-Dawley rats after autologous blood injection. Simvastatin or vehicle was administered orally at 1day after IVH and then daily for 1week. MRI studies were performed to measure the volumes of intracranial hematoma and lateral ventricle at days 1, 3, 7, 14, and 28 after IVH. Motor and neurocognitive functions were assessed at days 1 to 7 and 23 to 28, respectively. Iron deposition, iron-related protein expression, ependymal damage, and histology were detected at day 28. Expression of CD36 scavenger receptor (facilitating phagocytosis) was examined at day 3 after IVH using western blotting and immunofluorescence. Simvastatin significantly increased hematoma absorption ratio, reduced ventricular volume, and attenuated neurological dysfunction post-IVH. In addition, less iron accumulation and more cilia survival was observed in the simvastatin group when compared with the control. What's more, higher expression of CD36 was detected around the hematoma after simvastatin administration. Simvastatin significantly enhanced brain hematoma absorption, alleviated hydrocephalus, and improved neurological recovery after experimental IVH, which may in part by upregulating CD36 expression. Our data suggest that early simvastatin use may be a novel therapy for IVH patients.

Cell Surface CD36 Protein in Monocyte/Macrophage Contributes to Phagocytosis during the Resolution Phase of Ischemic Stroke in Mice

Infiltrating monocyte-derived macrophages (M-MΦ) influence stroke-induced brain injury. Although the inflammatory nature of M-MΦ in acute stroke has been well documented, their role during the resolution phase of stroke is less clear. With emerging evidence for the involvement of scavenger receptors in innate immunity, this study addresses an M-MΦ CD36 role in mediating phagocytosis during the recovery phase of stroke. Stroke increases CD36 and TSP-1/2 mRNA levels in the ipsilateral hemisphere at acute (3-day (d)) and recovery (7d) periods. Quantification of total, intracellular, and cell surface CD36 protein levels showed relatively unchanged expression at 3d post-ischemia. At 7d, there was a significant increase in cell surface CD36 (p < 0.05) with a concurrent reduction of intracellular CD36 (p < 0.05) in the ipsilateral hemisphere. Both cell surface and intracellular CD36 were found in whole brain lysates, whereas cell surface CD36 was predominantly detected in isolated brain mononuclear cells, blood monocytes, and peritoneal macrophages, suggesting that cell surface CD36 expressed in the post-ischemic brain originates from the periphery. The stroke-induced CD36 mRNA level correlated with increased expression of lysosomal acid lipase, an M2 macrophage marker. Functionally, higher CD36 expression in M-MΦ is correlated with higher phagocytic indices in post-ischemic brain immune cells. Moreover, pharmacological inhibition of CD36 attenuated phagocytosis in peritoneal macrophages and brain M-MΦ These findings demonstrate that cell surface CD36 on M-MΦ mediates phagocytosis during the recovery phase in post-stroke brains and suggests that CD36 plays a reparative role during the resolution of inflammation in ischemic stroke.

High-Fructose Intake Impairs the Hepatic Hypolipidemic Effects of a High-Fat Fish-Oil Diet in C57BL/6 Mice

Overnutrition of saturated fats and fructose is one of the major factors for the development of nonalcoholic fatty liver disease. Because omega-3 polyunsaturated fatty acids (n-3fa) have established lipid lowering properties, we tested the hypothesis that n-3fa prevents high-fat and fructose-induced fatty liver disease in mice. Male C57BL/6J mice were randomly assigned to one of the following diet groups for 14 weeks: normal diet (ND), high-fat lard-based diet (HFD), HFD with fructose (HFD+Fru), high-fat fish-oil diet (FOD), or FOD+Fru. Despite for the development of obesity and insulin resistance, FOD had 65.3% lower (P<0.001) hepatic triglyceride levels than HFD+Fru, which was blunted to a 38.5% difference (P=0.173) in FOD+Fru. The lower hepatic triglyceride levels were associated with a lower expression of lipogenic genes LXRα and FASN, as well as the expression of genes associated with fatty acid uptake and triglyceride synthesis, CD36 and SCD1, respectively. Conversely, the blunted hypotriglyceride effect of FOD+Fru was associated with a higher expression of CD36 and SCD1. During overnutrition, a diet rich in n-3fa may prevent the severity of hepatic steatosis; however, when juxtaposed with a diet high in fructose, the deleterious effects of overnutrition blunted the hypolipidemic effects of n-3fa.

Abstract 329: Microparticles Induce Antiangiogenic Signaling in Microvascular Endothelial Cells by Activating a CD36-Dependent Signaling Pathway Involving Src Kinases and Reactive Oxygen Species

Cell-derived microparticles (MP), ≤1μm vesicles shed from vascular and blood cells during activation or apoptosis, circulate in plasma and are biomarkers of cardiovascular disease. The type 2 scavenger receptor CD36 binds MP via phosphatidylserine exposed on their surface. On microvascular endothelial cells (MVEC) CD36 induces anti-angiogenic signals via its cognate protein ligands, such as thrombospondin-1. We hypothesized that CD36-MP interactions on MVEC would inhibit angiogenesis. MP generated in vitro from THP1 cells dose dependently inhibited MVEC tube formation in in vitro matrigel angiogenesis assays (&gt;50% inhibition of number of tubes formed, branch points, and segments; p&lt;0.05). MP derived from human umbilical vein endothelial cells (HUVEC), MVEC and erythrocytes also inhibited tube formation showing that the anti-angiogenic effect of MP is independent of their cell of origin. THP1 MP also inhibited MVEC migration in a transwell assay by 80% (p&lt;0.001). Early passage MVEC with high CD36 expression were inhibited more than late passage MVEC with low CD36 expression (80% vs 33%; p&lt;0.001) establishing the specificity of CD36 in this process. Further, in HUVEC that do not express CD36, inhibition of migration by THP1 MP was only 44% (p&lt;0.001) but lentiviral-mediated heterologous expression of murine CD36 in HUVEC restored inhibition of migration to levels similar to that of MVEC (82%; p&lt;0.001), corroborating the specificity of CD36 in MP mediated inhibition of migration. Since CD36 dependent generation of reactive oxygen species (ROS) is necessary to inhibit cell migration in macrophages, we tested if MP can induce ROS in MVEC. THP1 MP dose dependently induced ROS in MVEC by two fold compared to control at 1hr (p&lt;0.05). NADPH oxidase inhibitor, apocynin, blocked MP induced ROS induction by &gt;95% and partially (33%) reversed the inhibition of tube formation in MVEC by THP1 MP (p&lt;0.05). Western blots of THP1 MP treated MVEC showed time-dependent activation of Fyn kinase, and PP2, a Src family kinase inhibitor, blocked MP induced ROS induction by &gt;95%. These studies identify a novel CD36 dependent mechanistic pathway through which cell-derived MP inhibit MVEC angiogenic activities through ROS generation involving NADPH oxidase and Src kinase Fyn.

Microglial CX3CR1 required for M2 polarization and recovery after intracerebral hemorrhage (INC5P.322)

Abstract Intracerebral hemorrhage (ICH) occurs when a blood vessel in the brain ruptures, resulting in the activation of microglia. Upon activation, microglia polarize into a pro-inflammatory, M1 phenotype or an M2 phenotype associated with repair. Microglia and monocyte subsets express the chemokine receptor CX3CR1. CX3CR1-null microglia have been shown to have dysregulated inflammation. We hypothesize that microglial CX3CR1-deficiency promotes dysregulated, M1 microglia that do not aid in repair. C57BL/6 (WT) and CX3CR1GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with bone marrow from WT mice (CD45.1) to generate bone marrow chimeras (CD45.1→WT or CD45.1→CX3CR1-null). ICH was modeled by injecting 20μl of blood into the right striatum. Behavioral outcomes were measured using gait analysis, cylinder test, and beam walking. Mice were sacrificed 14 days after ICH; perihematomal regions were harvested for intracellular staining. The CD45.1→CX3CR1-null mice display greater neurological deficit 14 days after ICH by cylinder test (p=0.002), beam walking (p=&amp;lt;0.001) and gait analysis (p=0.02). By intracellular staining, WT microglia have produce more for IL-10 (p=0.002) and IL-4 (p=0.0003) than CX3CR1-null microglia. CX3CR1-null microglia have less SIRPα (p=0.02) and higher CD36 expression (p=0.0004) compared to WT microglia. Our results suggest CX3CR1 signaling is necessary for microglia to transition from M1 to M2 and contributes to recovery after ICH.

Lymphomonocyte subpopulations, activation and cytokine production in Alzheimer's Disease (AD)

AD is a neurodegenerative illness characterized by accumulation of β‐amyloid (A β‐42) and inflammation. To investigate the systemic signs of immune processes in AD we examined lymphomonocyte subpopulations (PBMCs) of 17 patients and 7 age‐matched healthy controls (HC). No differences were observed in T, B and NK subsets between the two groups, whereas an increase of both CD4+ and CD8+ naïve T cells was observed in AD patients. We analyzed AD and HC PBMCs after "in vitro" activation with Aβ‐42 and no differences were observed in the expression of early activation markers CD25 and CD69 on T and B cells, although AD cells show a basic activation. We also evaluated, on monocytes, the expression of the activation markers CD80 and HLA‐DR and of the receptor for fibrillar Aβ‐42, CD36. Here again no differences were observed between the two groups, even if there is a basic high expression of CD36 on AD cells. The evaluation of cytokines production after "in vitro" culture with Aβ‐42 shows an increased production of pro‐ and anti‐inflammatory cytokines and chemokines, in AD when compared to HC. These results, although puzzling, demonstrate an involvement of systemic immunity in AD patients.This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR, ex 60%)

CD36 (thrombospondin receptor) expression in childhood acute megakaryoblastic leukemia: In vitro drug sensitivity and outcome

The outcome for children with acute megakaryoblastic leukemia (AMKL) remains poor, except for cases associated with Down syndrome (DS). This study compared immunophenotypic and drug sensitivity patterns of childhood AMKL cases with or without DS. All DS-AMKL cases showed high expression of CD36 and were very sensitive to cytarabine and daunorubicin in vitro. In children without DS, high expression of CD36 on AMKL blasts identified a sub-group with immunophenotypic and drug sensitivity patterns similar to that of DS AMKL. Among non-DS AMKL patients, CD36-high cases had a superior outcome compared with CD36-negative cases. CD36 expression in acute myeloid leukemia cases other than AMKL was not associated with increased in vitro drug sensitivity. CD36 expression in AMKL may be an indicator of megakaryoblast maturation and chemotherapy sensitivity.