P2X7 and P2X4 mediate high glucose and palmitate-induced cell permeability.

<p>HUVEC monolayers were seeded in 24-well transwell inserts (0.4 μm) and exposed to high glucose and palmitate in the presence or absence of receptor antagonists for 48 h. 1 mg/ml FITC-dextran (MW 40,000 Da) was added in the upper well and the media collected from the lower well after 1 h. FITC-dextran flux was assessed by measuring the fluorescence at an excitation and emission wavelengths of 485 nm and 530 nm, respectively. Percent permeability was calculated and represented as fold difference relative to controls. n = 5 experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

Schematic representation of high glucose and palmitate-induced endothelial cell activation and dysfunction modulated in part by P2X7 and P2X4.

<p>Exposure of HUVECs to high glucose and palmitate results in the activation of the P2X7 and P2X4 causing (i) increased intracellular ROS and reduced eNOS contributing to endothelial cell dysfunction, and (ii) increased expression of IL-6, ICAM-1, VCAM-1, IL-8, and COX-2 resulting in endothelial cell activation. Blocking the P2X7 and P2X4 with AZ11645373 and PSB-12253, respectively, had a partial inhibitory effect on ROS as well as the inflammatory molecules with decreased leukocyte adhesion and vascular permeability. This is suggestive of the possible roles for P2X7 and P2X4 in modulating high glucose and palmitate-induced endothelial cell dysfunction, an early determinant of vascular disease.</p

P2X7 mediates high glucose and palmitate-induced increase in ICAM-1 & VCAM-1.

<p>qRT-PCR analysis shows increased <i>ICAM-1</i> (A) and <i>VCAM-1</i> (B) mRNA in response to high glucose and palmitate (24 h). The transcript levels were normalized to the housekeeping gene, <i>PPIA</i>. Representative epifluorescent images (x40 objective) of HUVECs exposed to high glucose and palmitate for 48 h show immunostaining for ICAM-1 (C; green) and VCAM-1 (D; red). The nuclei (blue) were visualized with NucBlue ready probe. Immunoblot analysis shows ICAM-1 (E; ~90 kDa)) and VCAM-1 (F) proteins normalized to β-Tubulin and are expressed as percentage of control with representative blots for each protein depicted. For VCAM-1, the antibody recognized multiple bands (a doublet in samples exposed to high glucose and palmitate and an additional band between 95–120 kDa), which were quantified and represented in the blot. n = 4 to 5 independent experiments each done in replicates; *<i>p</i> ≤ 0.05. Scale bar—20 μM.</p

P2X7 mediates high glucose and palmitate-induced increase in leukocyte adhesion.

<p>HUVEC monolayers seeded in 96-well plates were exposed to high glucose and palmitate in the presence or absence of receptor antagonists for 48 h. Leukocytes labeled with LeukoTracker were allowed to attach for 90 mins after which adherent cells were lysed and the fluorescence was measured at an excitation and emission wavelengths of 480 nm and 520 nm, respectively. n = 4 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

Knockdown of P2X7 and P2X4 inhibit high glucose and palmitate-induced expression of inflammatory genes.

<p>siRNA knockdown of P2X7 and P2X4 in the HUVECs inhibits high glucose and palmitate-induced (24 h) gene expression of <i>IL-1β</i> (A), <i>IL-6</i> (B), <i>PTGS2</i> (C), <i>IL-8</i> (D), <i>ICAM-1</i> (E), and <i>VCAM-1</i> (F). Cells transfected with the negative control siRNA (Neg. Ctl. siRNA) was used as controls and <i>PPIA</i> was used as the housekeeping gene to normalize all transcript levels. n = 3 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

P2X7 mediates high glucose and palmitate-induced ROS and eNOS.

<p>Intracellular ROS (A) in the HUVECs exposed to high glucose and palmitate for 24 h was measured with the fluorescent probe, H₂DCFDA. Immunoblot analysis of eNOS protein (B; 140 kDa) in HUVECs exposed to high glucose and palmitate for 48 h, which was normalized to β-Tubulin and represented as percentage of control. A representative blot for each protein is depicted. n = 5 to 6 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

High glucose and palmitate induces activation of p38-MAPK in a P2X7-dependent manner.

<p>HUVECs were exposed to high glucose and palmitate for 48 h and immunoblot analysis of cell lysates shows increased phosphorylated-p38-MAPK that was normalized to total p38-MAPK and expressed as percentage of control. A representative immunoblot for each protein is shown. n = 3 experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

Apyrase reduces high glucose and palmitate-induced expression of inflammatory genes.

<p>Apyrase reduces high glucose and palmitate-induced (24 h) expression of <i>IL-1β</i> (A), <i>ICAM-1</i> (B), and <i>VCAM-1</i> (C) in the HUVECs. The transcript levels normalized to <i>PPIA</i> are represented as fold change relative to the control. n = 3 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

P2X7 and P2X4 antagonists block high glucose and palmitate-induced expression of inflammatory genes.

<p>qRT-PCR analysis shows high glucose and palmitate-induced (24 h) transcript levels of <i>CASP1</i> (A), <i>IL-1β</i> (B), <i>IL-6</i> (C), <i>PTGS2</i> (D), and <i>IL-8</i> (E) in the presence or absence of the P2X7 (AZ11645373) and P2X4 (PSB-12253) antagonists. The transcript levels were normalized to the housekeeping gene, <i>PPIA</i>. n = 3 to 4 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p

Genotyping of Met-158-Thr in AMI cases and corresponding controls.

<p>The AMI case group (<i>n</i> = 1244) contains the early onset (EO), late onset (LO), family history (FH) and no family history (NFH) as well as early onset with family history (EO+FH) subgroups. *Both heterozygous and homozygous P2RY₁₃ Thr-158 carriers.</p