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. Author manuscript; available in PMC: 2016 Sep 12.
Published in final edited form as: Biorheology. 2015;52(5-6):447–463. doi: 10.3233/BIR-15067

Fig. 2.

Fig. 2

ANP inhibits PMN capture and shear resistant independent of cellular adhesion receptor activation. (A) PMN rolling, arrest, and transendothelial migration (TEM) quantified at 2 dynes/cm2 on IL-1β inflamed HUVEC was measured from video record as mean ± SEM number per four FOVs and analyzed from 3 separate microfluidic flow channels. n = 3 separate donors. * * * Signifies significance between ANP and vehicle control p < 0.005, ** signifies significance between ANP and vehicle control p < 0.01, and * signifies significance between ANP and control p < 0.05. (B) PMN (1 × 106 cells/ml) were pretreated with 1–10 nM ANP and continuously perfused over recombinant ICAM-1 + E-selectin substrates in a microfluidic flow chamber. The number of PMN remaining adherent relative to the number at 2 min under 2 dynes/cm2. Shear was incremented every 30 s as depicted up to 40 dynes/cm2. The numbers of PMN remaining in the presence or absence of ANP at the indicated dose were recorded from 3 separate donors. Nearly all PMN treated with vehicle control convert to shear resistant arrest, while ANP treated PMN formed long membrane tethers that abruptly ruptured upon detachment. Line plots are polynomial regression fits to the data points. (C) PMN tether formation for PMN arrested at 2 dynes/cm2 and ramped to 10 dynes/cm2. Images of tether length defined as point of contact with substrate to the center of the cell are shown just before cell detachment. PMN tether length significantly increased at the low and high dose of ANP treatment compared with vehicle control (p < 0.05, n = 3). (D) Isolated PMNs (1 × 106 cells/ml) were analyzed by flow cytometry for expression of β2-integrin and L-selectin adhesion receptors following 15 min stimulation with IL-8 (10 nM) after pretreatment with the indicated dose of ANP for 30 min prior to measurement. ANP did not alter the up-regulation of β2-integrin or the shedding of L-selectin when compared to untreated control (n = 3). PMN F-actin was measured using Phalloidin with and without IL-8 stimulation after simultaneous fixing and permeabilization. Data shows replicates for n = 3 donors with no significant effect of ANP observed.