Cellular Affinity Research Articles

Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

Abstract The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity.

Peripheral nerve regeneration through a silicone chamber implanted with negative carbon ions: Possibility to clinical application

Abstract We investigated whether a tube with its inner surface implanted with negative-charged carbon ions (C − ions) would enable axons to extend over a distance greater than 10 mm. The tube was found to support nerves regenerating across a 15-mm-long inter-stump gap. We also investigated whether a C − ion-implanted tube pretreated with basic fibroblast growth factor (bFGF) promotes peripheral nerve regeneration. The C − ion implanted tube accelerated nerve regeneration, and this effect was enhanced by bFGF. Silicone treated with C − ions showed increased hydrophilic properties and cellular affinity, and axon regeneration was promoted with this increased biocompatibility.

Multiple protein stationary phases: a review.

Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple binding sites, including orthosteric and allosteric sites. This review will address the utilization of immobilized cellular and tissue fragments to characterize multiple transmembrane proteins co-immobilized onto a stationary phase. This approach will be illustrated by demonstrating that multiple transmembrane proteins were immobilized from cell lines and tissue fragments. In addition, the immobilization of individual compartments/organelles within a cell will be discussed and the changes in the proteins binding/kinetics based on their location.

Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns

Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (−5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

Specific interactions between functionalised particles and circulating tumour cells.

Receptor-ligand binding has been one of the more popular approaches to specifically targeting tumour cells. In this work, targeting efficiency was quantitatively characterized using silica particles functionalized with EpCAM antibodies and EpCAM-expressing BT-20 breast cancer cells. The effects of incubation time and particle concentration on the number of functionalised particles bound to target cells were experimentally investigated. The number of bound particles was found to increase with particle concentration, but not necessarily with incubation time. Binding affinity loss because of cell-particle-cell interaction was identified as a limiting mechanism for the number of particles bound to target cells. While cell-surface coverage because of bound particles rises exponentially under low particle concentration, it features a peak value at high particle concentration. The current findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity.

In vitro biocompatibility evaluation of silk-fibroin/polyurethane membrane with cultivation of HUVECs

In order to investigate the in vitro biocompatibility of a novel polyurethane (PU) membrane modified by incorporation of superfine silk-fibroin powder (SFP), which was prepared for small-diameter vascular grafts, with the cultivation of human umbilical vein endothelial cells (HUVECs), PU and SFP were mixed with the ratios of 9:1, 7:3, 5:5, 3:7 (PU:SFP) to make four composite materials. Unmodified PU and polytetrafluoroethylene (PTFE) were added as control groups. CCK-8 assay was used to evaluate the cytotoxicity of these biomaterials. Data were processed using SPSS, and P < 0.05 was considered to be statistically significant. Adherence and spreading of HUVECs on the surface of specimens was observed using direct contact cultivation. The toxicity ratings of the novel composites were grade 0–1, which is in the acceptable range. In all the experimental groups except control, SFP/PU with ratio of 1:9 had the least cytotoxicity property, and more content of SFP in the composite showed no improvement of the biocompatibility. HUVECs strongly attached to and grew on the surface of the biomaterials, and proliferated rapidly. The proliferation ability increased with increased proportion of SFP; however the cell quantity on the surface of the materials decreased when the proportion of SFP was equal to or larger than that of PU in the composite. It is concluded that this novel material has excellent cellular affinity with no cytotoxicity to HUVECs. Adding SFP gives PU better biocompatibility, while further research on optimum blend ratios is still needed.

Polyhydroxyalkanoate Synthesis Affects Biosurfactant Production and Cell Attachment to Hydrocarbons in Pseudomonas sp. KA-08

Stressful conditions prevailing in hydrocarbon-contaminated sites influence the diversity, distribution, and activities of microorganisms. Oil bioremediation agents should develop special characteristics to cope with these environments like surfactant production and cellular affinity to hydrocarbons. Additionally, polyhydroxyalkanoate (PHA) accumulation was proven to improve tolerance to stressful conditions. Pseudomonas sp. KA-08 was isolated from a chronic oil-contaminated environment, it is highly tolerant to xylene, and it is able to accumulate PHA and to produce surfactant compounds that lower the water surface tension (ST) as well as bioemulsifiers. In this work, we studied the effect of the capability to accumulate PHAs on biosurfactant production and microbial attachment to hydrocarbons (MATH). Our results showed that PHA synthesis capability has a favorable effect in the production of compounds which affect the ST but not on the production of bioemulsifiers. On the other hand, PHA accumulation affects cellular affinity to xylene. MATH analysis showed that a PHA-negative mutant increased its affinity to xylene compared with the wild-type strain. This result was also observed in Pseudomonas putida GPp104 (a PHA(-) mutant), suggesting that this effect could be generalized to other Pseudomonas strains.

CD44-overexpressed gastric cancer imaging by near IR using HA-based supramolecular hydrogels.

34 Background: To establish NIR optical probe based on the HA-based supramolecular hydrogels (HASHs) conjugated with Cy5.5 for CD44-overexpressed gastric cancer imaging. Methods: To establish HASHs, Cy5.5 NHS ester was conjugated with polyethyleneimine (PEI, 25k Da) and mixed with hyaluronic acid (HA, 1M Da) by an electric interaction. The optimazed ideal molar ratio of PEI to HA was confirmed by DLS and gel electrophoresis. The CD44-expression level for various gastric cancer cell lInes (MKN1, MKN28, MKN45, MKN72, AGS, and N87 cells) was evaluated by FACS analysis. For establishment of the gastric cancer xenograft model, CD44-overexpressed gastric cancer cells were implanted into the BALB/c nude mouse's proximal thigh region. For in vitro targeting study, the cellular affinity of HASHs for CD44-low expressed gastric cancer cell line and CD44-overexpressed gastric cancer cell line was verified by confocal microscopy and IHC staing. For in vivo NIR imaging, HASHs were injected into established xenograft mouse via tail vein and NIR optical imaging was conducted time-dependently. Results: The colloidal size of HASHs was 1.4 micrometer and their morphology was confirmed by electron microscopy. CD44-expression level of MKN45 cells was 92.53% that was higher than MKN28 cells (2.66%). After the treatment of HASHs, the endocytosis into the cytosol was examined for MKN45 cells, but not observed in MKN28 cells due to the deficiency of CD44. 30 days after the transplantion of MKN45 cells, for in vivo imaging study, the prepared HASHs were intraveneously injected into tumor-bearing mouse model. By NIR optical imaging, the optical intensity at tumor site was enhanced upto 3 hours and the maximum intensity was 350 times larger than normal tissue. Conclusions: HASHs was established using supramolecular HA and Cy5.5-conjugated PEI for the targeted imaging of CD44-overexpressed gastric cancer cells. In vitro and in vivo studies demonstrated that SHAHs can visualize the individualized CD44-overexpressed gastric cancer cells by non-invasive optical imaging.

Photophysical Properties of Pheophorbide-a Derivatives and Their Photodynamic Therapeutic Effects on a Tumor Cell LineIn Vitro

Pheophorbide-a derivatives have been reported to be potential photosensitizers for photodynamic therapy (PDT). In this study, photophysics of pheophorbide-a derivatives (PaDs) were investigated along with their photodynamic tumoricidal effectin vitro. PaDs were modified by changing the coil length and/or making the hydroxyl group (–OH) substitutions. Their photophysical properties were studied by steady-state and time-resolved spectroscopic methods. The photodynamic tumoricidal effect was evaluated in the mouse breast cancer cell line (EMT6). Lifetime and quantum yield of fluorescence and quantum yields of triplet state and singlet oxygen were studied to determine the dynamic energy flow. The coil length of the substituted alkyl group did not significantly affect the spectroscopic properties. However, the substitution with the hydroxyl group increased the quantum yields of the triplet state and the singlet oxygen due to the enhanced intersystem crossing. In order to check the application possibility as a photodynamic therapy agent, the PaDs with hydroxyl group were studied for the cellular affinity and the photodynamic tumoricidal effect of EMT6. The results showed that the cellular affinity and the photodynamic tumoricidal effect of PaDs with the hydroxyl group depended on the coil-length of the substituted alkyl group.

Effect of selective oxidation of bacterial cellulose on degradability in phosphate buffer solution and their affinity for epidermal cell attachment

Oxidized bacterial cellulose showed the 3D nano-fibrils structure of BC. The mass loss and degradation rate of OBC were much higher than those of BC. When immersed in PBS, OBC degraded gradually. Cell-adhesion and proliferation studies revealed that OBC had excellent cellular affinity.

Enhanced Cell Affinity of Chitosan Membranes Mediated by Superficial Cross-Linking: A Straightforward Method Attainable by Standard Laboratory Procedures

It is well accepted that the surface modification of biomaterials can improve their biocompatibility. In this context, techniques like ion etching, plasma-mediated chemical functionalization, electrospinning, and contact microprinting have successfully been employed to promote the cell adhesion and proliferation of chitosan (CH) substrates. However, they prove to be time-consuming, highly dependent on environmental conditions, and/or limited to the use of expensive materials and sophisticated instruments not accessible to standard laboratories, hindering to a high extent their straightforward application. Filling this gap, this paper proposes the superficial cross-linking of CH as a much simpler and accessible means to modify its superficial properties in order to enhance its cellular affinity. CH membranes were prepared by solvent casting followed by a cross-linking step mediated by the chemical vapor deposition (CVD) of glutaraldehyde (GA). The membranes were characterized against non- and solution cross-linked membranes in terms of their mechanical/surface properties and biological performance. Among others, the CVD membranes proved (i) to be more mechanically stable against cell culture and sterilization than membranes cross-linked in solution and (ii) to prompt the adherence and sustained proliferation of healthy cells to levels even superior to commercial tissue culture plates (TCPs). Accordingly, the CVD cross-linking approach was demonstrated to be a simple and cost-effective alternative to the aforementioned conventional methods. Interestingly, this concept can also be applied to other biomaterials as long as GA (or other volatile components alike) can be employed as a cross-linker, making possible the cross-linking reaction at mild experimental conditions, neither requiring sophisticated lab implements nor using any potentially harmful procedure.

Continuous glucose monitoring during early phase of acute ischemic stroke in patients without diabetes mellitus history

Polylactide-co-glycolic acid(PLGA) tissue engineering scaffolds play an important role in the regeneration of bone and cartilage.Due to the poor hydrophilicity of PLGA,it is difficult for cells to attach to the scaffolds.Modification by RGD(Arg-Gly-Asp) peptides can effectively improve the cellular affinity of PLGA and adhesion and proliferation of the seed cells.This review summarizes the recent progress in PLGA tissue engineering scaffolds modified by RGD peptides.

Abstract C224: Identification of drug targets via their intracellular interactions.

Abstract Phenotypic-based screening of small molecule libraries is a significant trend in drug discovery. However, deciphering the intracellular interactions between proteins and small molecules that mediate phenotypic outcomes remains a major challenge. Current approaches rely on linking the small molecule to a surface or affinity tag (e.g., biotin), allowing selective capture of the corresponding protein target for identification by mass spectrometry. Verifying that the linkage method does not disrupt the pharmacological activity is important for ensuring maintenance of the binding interaction with the intracellular targets. Consequently, although target capture is commonly done from cell lysates, methods compatible with living cells are preferable. We have developed such a method based on a novel chloroalkane tag that minimally affects compound potency and cell permeability. This allows phenotypic outcomes to be recapitulated by the modified compound, increasing confidence in the biological relevance of the captured proteins. The tagged compound is allowed to bind at equilibrium to intracellular targets, then cells are lysed and the chloroalkane bound to its protein targets is quickly captured onto magnetic particles containing immobilized HaloTag. The rapid isolation method minimizes complex collapse, preserves low affinity interactions, and in combination with low background adsorption facilitates target identification by mass spectrometry. Engagement of identified targets by compounds in living cells can be verified using bioluminescence energy transfer (BRET). NanoLuc luciferase fused to the target protein serves as a BRET donor to the compound derivatized with fluorophore adducts. As a model to test this combined target capture/target validation work flow we selected the interaction of histone deacetylase (HDAC's) and Vorinostat (SAHA) a broad- HDAC inhibitor. This family of deacetylases exhibits a range of cellular abundance and affinity to SAHA (0.001-1µM), and has members localized in the nucleus or cytoplasm. Results: Treatment of K562 cells with SAHA or a derivative having the chloroalkane tag revealed high cellular potency against HDAC class I/IIb with IC50 of 0.1µM and 0.2µM respectively. These results indicate minimal impact of the chloroalkane modification on the drug potency. For target identification, the SAHA chloroalkane derivative was allowed to bind at equilibrium to intracellular targets followed by rapid capture onto immobilized HaloTag. By this method we were able to identify all the direct interactors of SAHA (i.e., HDAC 1,2,3,6 and 8) regardless of their subcellular localization, abundance, or affinity. The intracellular BRET approach was next used to validate the set of identified HDAC's and to rank order their relative binding affinity to SAHA. Our results suggested that these approaches used in combination provide reliable identification of targets in living cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C224. Citation Format: Rachel Friedman Ohana, Matt Robers, Thomas A. Kirkland, Carolyn C. Woodroofe, Chad Zimprich, Tetsuo H. Uyeda, Paul Otto, Sergiy Levin, Keith Wood. Identification of drug targets via their intracellular interactions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C224.

Interview with Irving W Wainer

Irving W Wainer, Senior Investigator in the Intramural Research Program at the National Institute on Aging/NIH received his PhD degree in chemistry from Cornell University and did postdoctoral doctoral studies in molecular biology (University of Oregon) and clinical pharmacology (Thomas Jefferson Medical School). He worked for the US FDA and held positions at St Jude's Children's Research Hospital, at McGill University as Professor in the Department of Oncology, and as a Professor of Pharmacology at Georgetown University. Wainer has published over 350 scientific papers, 10 books, 25 book chapters and holds 11 patents. His awards include: 'A.J.P. Martin Medal' presented by the Chromatographic Society; Doctor HonorisCausa awarded by the Medical University of Gdansk (Gdansk, Poland, 2006), Doctor HonorisCausa awarded by the Department of Medicine, University of Liege (Liege, Belgium, 2012), and the 2013 Eastern Analytical Symposium Award for Outstanding Contributions to the Fields of Analytical Chemistry. Wainer's research includes the development of new therapeutic agents for the treatment of congestive heart failure, cancer, pain and depression, many of which are in the later stages of drug development. His laboratory has also continued the development of cellular membrane affinity chromatography technology, and recent work includes the development of columns containing immobilized forms of the breast cancer resistance protein found in cellular and nuclear membranes and mitochondrial membrane columns. Wainer's laboratory has also continued its study of the effect of disease progression and aging on drug metabolism in critically ill and terminal patients. Interview was conducted by Lisa Parks, Assistant Commissioning Editor of Bioanalysis.

Functional characterization of the parathyroid hormone 1 receptor in human periodontal ligament cells

Intermittent parathyroid hormone (PTH) exerts anabolic effects on bone and has been approved for osteoporosis therapy. The dual actions of PTH are mediated primarily through the parathyroid hormone 1 receptor (PTH1R). Upon ligand binding, PTH1R activates diverse signaling pathways, including cAMP/protein kinase A (PKA)- and phospholipase C/protein kinase C (PLC/PKC)-dependent pathways. PTH1R has been abundantly studied in bone cells. Knowledge on PTH1R characteristics and physiology in periodontal ligament (PDL) cells is still in its infancy. We characterized PTH1R in PDL cells in terms of its cellular localization, binding affinity, and signal transduction and compared these characteristics to those of MG63 osteoblast-like cells. PTH1R mRNA/protein was identified in PDL and MG63 cells. PTH1R was mainly localized on the plasma membrane, in vesicular structures inside the cell, and, to some extent, in the nucleus of both cell types. Binding characteristics of PTH1R were cell type specific, with PDL cells demonstrating a lower binding affinity. The response of cAMP and active PKC production in MG63 cells was dose dependent with increasing PTH(1-34) concentration, whereas in PDL cells, it was regulated biphasically. However, we observed a cross talk between the cAMP/PKA and PLC/PKC signaling pathways, which were regulated diametrically opposed at a given concentration of PTH(1-34). These data indicate that, albeit the similarity in its subcellular distribution, PTH1R in PDL cells exhibits characteristics different from those in MG63 cells, pointing to the cell type specificity of this receptor. The findings further elucidate the characteristics of PTH action in dental tissues and widen the theoretical basis for the development of anabolic treatment strategies.

The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells

BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC–MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

In vitro bioactivity evaluation of nano- and micro-crystalline anodic TiO2: HA formation, cellular affinity and organ culture

Anodization is an easily viable technique useful for producing TiO2 coatings on titanium substrates. Nano-crystalline anodic TiO2 structure was produced on titanium at 20V using 1M Na2SO4 and 0.5% NaF and consolidated by a further heat-treatment. Micro-crystalline anodic TiO2 was produced on titanium by applying a galvanostatic current density of 70A/m2 in water medium. To assess the usefulness of these nano- and micro-oxides for bone implant stability, physical properties and bone in vitro bioactivity including HA formation, cellular affinity and mouse-tissue morphogenesis, were evaluated. Bioactivity of the different anodic surfaces was evaluated by treating them in a simulated body fluid (SBF) to form hydroxyapatite (HA) and the rates of HA formation were compared. Deposits of HA could be seen on the nano-oxide surface within 7days, whereas HA was detected only after 14days on the micro-oxide surface. In vitro cell culture tests done using mouse osteoblasts indicated that the nano-oxides showed statistically significant cell activity than the micro-oxides and the machined titanium. Branching morphogenesis test done for 72h on these surfaces showed more branching on the micro- and nano-oxides as compared with titanium surface.

A novel method for the production and evaluation of hernia repair mesh in an in vitro environment

While most hernia mesh researchers use animal models or patient studies to evaluate prostheses, we have developed an in vitro-centric mesh production and examination system to characterize mesh properties that affect the cellular affinity and treatment potential of repair mesh. A loom was designed and constructed, and a feasibility study was conducted on auto-loom woven, degummed silk mesh seeded with murine-derived D1 multipotent mesenchymal stromal cells (MSCs). Meshes of two different pore sizes, 1 mm and 2 mm, were investigated. The seeded meshes were evaluated using a variety of assays to determine cellular metabolic activity, viability, and conformation at time points of 7, 14, and 21 days. The assays showed substantial cell attachment to the mesh at all time points, including penetration of cells into the interior of silk threads. Metabolic activity peaked at Day 14 for both mesh sizes, with a sharp rise in protein production from Day 7 to 14. Based upon the successful production of mesh and acquisition of results from the assays performed, it can be concluded that an in vitro evaluation system for hernia mesh is both possible and likely to produce scientifically relevant results.

Controlled heparin conjugation on electrospun poly(ε-caprolactone)/gelatin fibers for morphology-dependent protein delivery and enhanced cellular affinity

Electrospun fibrous scaffolds have now been shown to possess great potential for tissue engineering applications, owing to their unique mimicry of natural extracellular matrix structure. In this study, poly(ε-caprolactone) and gelatin were electrospun to fabricate tissue-engineered scaffolds with three different fiber morphologies (1.0μm, 3.0μm and co-electrospun containing both 1.0 and 3.0μm diameter fibers). Subsequently, these scaffolds were conjugated with heparin to immobilize a bioactive molecule by electrostatic interactions. This study determined the quantity of heparin conjugation on the scaffolds and that the crosslinking time and the fiber morphologies govern the extent of heparin conjugation on the fibers. In order to evaluate the release capacity of the heparin-conjugated scaffolds, lysozyme was used as a model protein for conjugation. The heparin-conjugated scaffolds provided high loading efficiency and cumulative release of lysozyme with a relatively linear relationship. In addition, the release kinetics was significantly dependent on heparin conjugation and fiber morphology. This fundamental investigation into how fiber morphology and crosslinking protocols can affect the heparin binding ability of electrospun fibers is crucial for predicting the delivery of many different types of bioactive molecules from an electrospun scaffold for tissue engineering applications.

CD36 Ectodomain Phosphorylation Blocks Thrombospondin-1 Binding

Objective— CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. We determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein kinase C (PKC) in melanoma cells. Methods and Results— Phosphorylation of CD36 peptide on Thr92 by PKCα suppressed binding of thrombospondin-1 peptides in vitro, and the level of inhibition correlated with the level of phosphorylation. Basal phosphorylation levels of CD36 in vivo in platelets, endothelial cells, and melanoma cells were assessed by immunoprecipitation and immunoblot and were found to be very low. Treatment of CD36-transfected melanoma cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand-mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface–associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation. Conclusion— New protein synthesis and trafficking through the Golgi are required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly. These studies suggest a novel in vivo pathway for CD36 phosphorylation that modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling.