Elevated arginase1 expression and increased arginase inhibitor-induced relaxation of Cavin-1^−/− arteries.

<p>A and B) Western blotting for arginase 1 in small mesenteric arteries (SMA) A) and in aorta B) from wild type (WT) and cavin-1 knock out (KO) mice. C) SMA were mounted in a wire-myograph and pre-contraction with 40 mM KCl. Relaxation to increasing concentrations of NOHA, an arginase inhibitor, was then assessed.</p

Unchanged mean arterial blood pressure in cavin-1-deficient mice.

<p>Mean arterial blood pressure was measured in the carotid artery of anaesthetized mice in control conditions (A) and after infusion of the nitric oxide synthase inhibitor L-NAME (B). Panel C shows heart rate in control conditions.</p

Cavin-1-reporter staining of systemic blood vessels.

<p>A) The aorta was dissected, fixed briefly in paraformaldehyde, and stained using x-gal. Bottom shows an intact aorta and top shows an opened aorta from the luminal side. B) Small mesenteric arteries with the superior mesenteric artery on the left. C) The vascular supply of the cerebral hemispheres. D) X-gal stained arteries and a vein in the prostate was paraffin embedded, sectioned, and counterstained using hematoxylin-eosin.</p

Cavin-1 deficient mice exhibit reduced vasomotion.

<p>Panel A) shows original traces from two wild type (WT; black) and two knock out (KO; grey) arteries, mounted in a wire-myograph, after prolonged (1 h) incubation with cirazoline (0.1 μM). Note the absence of rhythmic force oscillations (vasomotion) in KO. Panel B) shows the fraction of vessels exhibiting vasomotion in WT and KO in the presence and absence of the gap junction blocker 18-α-glycyrrhetinic acid (18-α-GA). Panel C) shows expression of connexin 40 and 43 in the aorta and D) shows that 18-α-GA does not reduce the difference between WT and KO in α₁-adrenergic contraction.</p

Reduced expression of caveolae-associated proteins in cavin-1^−/− mice.

<p>A) 20 μg of protein from small mesenteric artery lysates was subjected to western blotting for cavins and caveolins. GAPDH or HSP90 were used as loading controls. B) Electron micrographs showing membrane sections from wild type (WT) and cavin-1-deficient (KO) small mesenteric arteries. Top panels show endothelial cells (EC) and bottom panels show smooth muscle cells (SMC). Middle panel in B shows quantification of caveolae per μm membrane in endothelial cells (EC) and in smooth muscle cells (SMC) from WT and KO animals, respectively. Right panel shows size distribution of caveolae in endothelium compared to smooth muscle in WT arteries.</p

Increased α₁-adrenergic contraction in small mesenteric arteries from cavin-1-deficient mice.

<p>A) Original traces showing contraction to 60 mM KCl and to cumulative addition of cirazoline in one representative wild type (WT; black) and knock out (KO; grey) mouse. Concentration-response curves for the α₁-adrenergic agonist cirazoline (C, control conditions; D, +300 μM of the NOS-inhibitor L-NAME; E, in the presence of 20 μM ODQ; F, in the presence of 10 μM ryanodine; G, in the presence of 20 μM dynasore) in small mesenteric arteries shows increased α₁-adrenergic contractility in the absence of cavin-1. As a reference, contraction to cirazoline in WT and KO is replicated in grey in panel D, E, F and G. The statistical difference between WT-drug and KO-drug is indicated by the p-value in the graphs. Panel B shows integrated force in response to depolarization (60 mM KCl).</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

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

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 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