American Society of Hematology

Figure 1

$Figure 1. LPA down-regulates CD36 expression in human and mouse MVECs. (A) Representative flow cytometry histogram (left) of human MVECs after exposure in complete medium to LPA (50μM), PMA (100 ng/mL), or vehicle control (CTL) for 36 hours. Cells were analyzed with FITC-conjugated anti-CD36 IgG. An isotype-matched nonimmune IgG was used as a control (brown). On the right is a bar graph showing means ± SD of CD36 median fluorescence intensity for triplicate experiments (P < .05). (B) Time course (left) and dose response (right) of CD36 down-regulation in MVECs exposed to LPA. The time course was done using 5μM LPA, and dose response was assessed at 24 hours. In both cases cells were grown first to 90% confluence and then incubated overnight in “basic” medium containing 1% FBS before addition of LPA. The inset shows a representative blot. The bar graph for the time course shows means ± SEM of band densities (normalized to β-actin control) for 3 independent experiments. The 48-hour bar on the time course indicates relative expression 48 hours after initiation of LPA exposure; in this experiment the LPA-containing medium was removed after 24 hours and replaced with complete medium for an additional 24 hours. The dose response (right panel) was determined by flow cytometry as in panel A. Mean fluorescence intensities relative to the isotype control were obtained for each treatment, and the fluorescence intensity from cells not exposed to LPA was set as 100. (C) Membrane fractions of human MVECs exposed to LPA were obtained by differential centrifugation and then analyzed by Western blot for CD36 expression as in panel B. (D) Quantitative real-time PCR for CD36 mRNA from HMVECs exposed to LPA (5μM) or S1P (1μM) for timed periods from 8 to 24 hours. Data are expressed relative to a control transcript, GAPDH and the mean value for untreated samples were set to 1, and the bar graph shows mean ± SEM of 3 independent experiments. (E) CD36 mRNA transcription (nuclear run-on) assays performed on nuclei isolated from human MVECs treated with LPA (5μM) or vehicle control for 24 hours. Nuclei were incubated with ATP, CTP, GTP, and biotin-16-UTP (2.5mM) at 30°C for 45 minutes, and the biotinylated transcripts were then purified and used for cDNA synthesis and real-time PCR. Data are presented as relative mRNA levels of CD36 compared with GAPDH and are mean ± SEM from n = 3. (F) Mouse cardiac MVEC CD36 protein expression detected by Western blot of whole-cell lysates 24 hours after exposure to LPA (1-5μM). Blots were performed as in panel B using anti-CD36 antibody and then stripped and reprobed with anti–β-actin as a loading control. The gel is representative of 3 separate experiments, and numbers below it show the mean band density ± SD compared with the control lane.$

LPA down-regulates CD36 expression in human and mouse MVECs. (A) Representative flow cytometry histogram (left) of human MVECs after exposure in complete medium to LPA (50μM), PMA (100 ng/mL), or vehicle control (CTL) for 36 hours. Cells were analyzed with FITC-conjugated anti-CD36 IgG. An isotype-matched nonimmune IgG was used as a control (brown). On the right is a bar graph showing means ± SD of CD36 median fluorescence intensity for triplicate experiments (P < .05). (B) Time course (left) and dose response (right) of CD36 down-regulation in MVECs exposed to LPA. The time course was done using 5μM LPA, and dose response was assessed at 24 hours. In both cases cells were grown first to 90% confluence and then incubated overnight in “basic” medium containing 1% FBS before addition of LPA. The inset shows a representative blot. The bar graph for the time course shows means ± SEM of band densities (normalized to β-actin control) for 3 independent experiments. The 48-hour bar on the time course indicates relative expression 48 hours after initiation of LPA exposure; in this experiment the LPA-containing medium was removed after 24 hours and replaced with complete medium for an additional 24 hours. The dose response (right panel) was determined by flow cytometry as in panel A. Mean fluorescence intensities relative to the isotype control were obtained for each treatment, and the fluorescence intensity from cells not exposed to LPA was set as 100. (C) Membrane fractions of human MVECs exposed to LPA were obtained by differential centrifugation and then analyzed by Western blot for CD36 expression as in panel B. (D) Quantitative real-time PCR for CD36 mRNA from HMVECs exposed to LPA (5μM) or S1P (1μM) for timed periods from 8 to 24 hours. Data are expressed relative to a control transcript, GAPDH and the mean value for untreated samples were set to 1, and the bar graph shows mean ± SEM of 3 independent experiments. (E) CD36 mRNA transcription (nuclear run-on) assays performed on nuclei isolated from human MVECs treated with LPA (5μM) or vehicle control for 24 hours. Nuclei were incubated with ATP, CTP, GTP, and biotin-16-UTP (2.5mM) at 30°C for 45 minutes, and the biotinylated transcripts were then purified and used for cDNA synthesis and real-time PCR. Data are presented as relative mRNA levels of CD36 compared with GAPDH and are mean ± SEM from n = 3. (F) Mouse cardiac MVEC CD36 protein expression detected by Western blot of whole-cell lysates 24 hours after exposure to LPA (1-5μM). Blots were performed as in panel B using anti-CD36 antibody and then stripped and reprobed with anti–β-actin as a loading control. The gel is representative of 3 separate experiments, and numbers below it show the mean band density ± SD compared with the control lane.

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