2FF inhibits microvascular stasis and leukocyte rolling and adhesion in sickle mice in vivo.

(A) NY1DD transgenic sickle mice (n = 4/group) were given the indicated concentration of 2FF in their drinking water for 7 days. Microvascular stasis was measured on day 7, one hour after the infusion of stroma-free Hb (3.2 μmols heme/kg). *P≤.05 for all pairwise comparisons. (B) NY1DD sickle mice were gavaged with water or 150 mg/ml 2FF (.01 ml/g, twice per day) for 1 or 3 days. After the indicated 2FF treatment period, stroma-free Hb (3.2 μmols heme/kg body weight) was infused intravenously and microvascular stasis was measured 1 hour later. *P≤.01 for water vs 2FF. (C) NY1DD and HbSS-Townes sickle mice (n = 3/group) were given 100 mM 2FF in their drinking water for 7 days. Stasis was measured on day 7 after exposure to 1h of hypoxia and 1h of reoxygenation (H/R). *PD) Leukocyte rolling flux was measured in venules in the DSFC windows of NY1DD mice before (baseline) and 1h after infusion of Hb (3.2 μmol/kg). Half of the mice (n = 4) were treated with 2FF (100 mM in drinking water X 7 days) prior to baseline measurements. Control mice (n = 4) were given drinking water without 2FF. The rolling flux was determined as the total number of leukocytes rolling through a given section of vessel per minute. Values are means + SD. #P*pE) Leukocyte adhesion was measured in the same venules as described in (D). Values are mean number of adherent cells per 100 μm + SD. #P*pF and G) PMNs (F) and SS-RBCs (G) were isolated from HbSS-Townes mice (n = 2 mice twice) given drinking water or 100 mM 2FF in drinking water for 7 days. Fluorescently labeled PMNs or SS-RBCs were incubated with resting and activated HUVEC (4 wells/treatment) for 30 minutes. Activated HUVEC, which express P-selectin on their surface, were prepared by incubating HUVEC with 10 μM heme for 30 minutes. Values are mean % area of HUVEC cell surface covered by PMNs (F) or SS-RBCs (G) + SD after HUVEC washing. #P*p