Objective Based on previous evidence that polymerase delta interacting protein 2 (Poldip2) increases NADPH oxidase 4 (Nox4) activity in vascular smooth muscle cells (VSMC), we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species (ROS) production and alter vascular function. Approach and Results Because homozygous Poldip2 deletion is lethal, Poldip2+/− mice were employed. Poldip2 mRNA and protein levels were reduced by about 50% in Poldip2+/− aorta, with no change in p22phox, Nox1, Nox2 and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2+/− tissue. Isolated aortas from Poldip2+/− mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness and reduced compliance, associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured VSMC from Poldip2+/− mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2+/− mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. Conclusions Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.

BACKGROUND Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow. STUDY DESIGN AND METHODS AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled. RESULTS RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging. CONCLUSIONS These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.

The contribution of medial calcification to vascular dysfunction in renal failure is unknown. Vascular function was measured ex vivo in control, noncalcified uremic, and calcified uremic aortas from rats with adenine-induced renal failure. Plasma urea was 16 ± 4, 93 ± 14, and 110 ± 25 mg/dl, and aortic calcium content was 27 ± 4, 29 ± 2, and 4,946 ± 1,616 nmol/mg dry wt, respectively, in the three groups. Maximal contraction by phenylephrine (PE) or KCl was reduced 53 and 63% in uremic aortas, and sensitivity to KCl but not PE was increased. Maximal relaxation to acetylcholine was impaired in uremic aortas (30 vs. 65%), and sensitivity to nitroprusside was also reduced, indicating some impairment of endothelium-independent relaxation as well. None of these parameters differed between calcified and noncalcified uremic aortas. However, aortic compliance was reduced in calcified aortas, ranging from 17 to 61% depending on the severity of calcification. We conclude that uremic vascular calcification, even when not severe, significantly reduces arterial compliance. Vascular smooth muscle and endothelial function are altered in renal failure but are not affected by medial calcification, even when severe.

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