Fig 1.
Incubation of human saphenous vein endothelial cells (HSVEC) in Normal Saline but not Plasma-Lyte leads to loss of membrane integrity.
HSVEC were incubated in basal medium (Ctrl), Normal Saline (NS) or Plasma-Lyte (PL) for 2 hours, fixed, and stained with phalloidin (A, red stain; scale bar = 5μm). Supernatants were collected and LDH activity (B) and ATP levels (C) were measured (*p < 0.05, n = 3–5, where n = the number of cell plates from different passages of cells). Transendothelial electrical resistance (TEER) (D) was measured 2 hours after treatment with NS with an epithelial voltohmmeter using STX2 chopstick electrodes (*p < 0.05, number of cell plates (n) = 6 in triplicates from different passages of cells).
Fig 2.
Incubation in Normal Saline solution but not Plasma-Lyte leads to impaired endothelial dependent relaxation of rat aorta and human saphenous vein.
Rings of rat aorta (RA, n = 5–17 rats) were suspended in the muscle bath (Ctrl) or incubated for 2 hours in NS or PL in the presence or absence of 2’(3’)-O-(Benzoylbenzoyl) adenosine 5’–triphosphate (BzATP, 500μM); ATP (20mM), apyrase (Apy, 4 units/ml), A740003 (A74, 100 μM), or SB203580 (SB, 20 μM) (A). Rings of human saphenous vein (HSV, n = 5–6 patients) were suspended in the muscle bath (Ctrl) or incubated for 2 hours in NS in the presence or absence of apyrase (Apy, 4 units/ml) or SB203580 (SB, 20 μM) (B). The strips were suspended in a muscle bath and pre-contracted with phenylephrine (5 x 10−7M) and treated with carbachol (5 x 10−7 M), *p < 0.05.
Fig 3.
Stimulation of human saphenous vein endothelial cells (HSVEC) leads to activation of p38 MAPK/MAPKAP kinase 2; IL-1β release; and increased VCAM expression.
HSVEC (n = 4–5 duplicate plates from different passages of cells) were incubated in growth medium (Ctrl), Normal Saline (NS) or Plasma-Lyte (PL) for 2 hours. The cells were lysed, lysates separated and immunoblotted for activated (phosphorylated, p-p38 MAPK) and total p38MAPK (A) or activated (phosphorylated, p-MK2) and total MK2 (B). Representative immunoblot and cumulative data of relative phosphorylation compared to untreated cells are shown (*p < 0.05). For IL-1β expression, HSVEC were primed with TNF (10ng/ml) and IFN (100ng/ml) for 48 hours and then treated with ATP (3mM) for 2 hours in the presence or absence of oATP (300μM), A438079 (A43; 50μM), or SB203580 (SB, 10μM), or MK2 inhibitor (MK2i, 100μM) for 24 hours and IL-1β was measured in the medium using an ELISA kit (C, *p < 0.05, n = 5–6). Cells were treated with ATP (1mM) for 6 hours in the absence or presence of A43 (50μM) and lysates prepared and immunoblotted for VCAM levels (D, *p < 0.05, n = 5–6 plates of cells from different passages).
Fig 4.
Treatment with IL-1β impairs endothelial-dependent relaxation in rat aorta (RA), activates p38MAPK and MK2 and increases arginase II activity in HSVEC.
(A) Rings of RA (n = 12–20 rats) were suspended in the muscle bath and left untreated (Ctrl), treated with IL-1β (10, 50, and 100ng/ml), or co-treated with IL-1β (100ng/ml) and SB203580 (20μM) for 3 hours. Tissues were pre-contracted with phenylephrine (5x10−7M) and treated with carbachol (5x10-7 M) and endothelial relaxation was measured, *p < 0.05, n = 12–20 rats. HSVEC were either untreated (Ctrl) or treated with IL1-β (10 ng/ml) for the times indicated. Cells were lysed, separated and immunoblots were probed for activated (phosphorylated, p-p38MAPK) or total p38MAPK (B), activated (phosphorylated p-MK2) or total MK2 (C), and arginase II (ArgII, D). Representative immunoblots and cumulative data of relative phosphorylation compared to untreated cells shown. *p < 0.05, n = 4–5.
Fig 5.
Incubation in Normal Saline (NS) or Plasma-Lyte (PL) rendered acidic leads to decreased endothelial dependent relaxation in intact naïve porcine saphenous veins (PSV).
Rings of PSV were incubated in PL; NS; or PL in which the pH had been adjusted to 5.5 or 6.0 for 2 hours. The strips of PSV were then suspended in a muscle bath and pre-contracted with phenylephrine (5 x 10−7M) and treated with carbachol (5 x 10−7 M, n = 5–7 pigs * p < 0.05).
Fig 6.
Resuscitation with Normal Saline but not Plasma-Lyte leads to acidosis and endothelial dysfunction in a porcine model.
A drop in bicarbonate (A, *p < 0.05, n = 3 pigs in each group) arterial base excess (ABE) (B *p < 0.05, n = 3 pigs in each group) occurred with 2500 mL and 5000 mL, and a drop in pH occurred with 5000 mL (C, * p < 0.05, n = 3 pigs each group) of NS administration. No significant changes occurred during whole blood removal, whole blood return, or PL administration. PSV were atraumatically harvested following resuscitation with NS or PL and suspended in a muscle bath then pre-contracted with phenylephrine (5 x 10−7 M) and treated with carbachol (D, 5 x 10−7 M, n = 3 pigs in each group, * p < 0.05, unpaired t-test).
Fig 7.
Model of molecular mechanisms of vascular injury to endothelium by ATP.
Injury to vascular tissues (including storage of vein graft in Normal Saline) leads to membrane disruption and ATP release. Extracellular ATP activates the P2X7R/p38MAPK pathway, leading to IL-1β production and inflammation. Activation of P2X7R leads to increased extracellular ATP via release from P2X7R channels/pores. Prolonged exposure to IL-1β leads to increased arginase II expression and activation of P38MAPK, which contributes to endothelial dysfunction and amplification of the response to injury.