A(3) adenosine receptor activation during reperfusion reduces infarct size through actions on bone marrow-derived cells

Abstract

The goal of this study was to examine whether the A(3) adenosine receptor (A(3)AR) agonist Cl-IB-MECA protects against myocardial ischemia/reperfusion injury when administered at the time of reperfusion in an in vivo mouse model of infarction induced by 30min of coronary occlusion and 24h of reperfusion. Treating B6 wild-type with Cl-IB-MECA during the reperfusion phase (100microg/kg i.v. bolus+0.3microg/kg/min subcutaneously via implantation of Alzet mini-osmotic pumps) reduced myocardial infarct size approximately 37% from 50.1+/-2.5% in vehicle-treated mice to 31.6+/-2.8% in Cl-IB-MECA-treated mice, and significantly reduced the number of leukocytes that infiltrated into the ischemic-reperfused myocardium. Cl-IB-MECA did not reduce infarct size or limit leukocyte accumulation in studies using B6 congenic A(3)AR gene "knock-out" mice or in chimeric mice lacking the expression of A(3)ARs in bone marrow (BM)-derived cells. Subsequent mechanistic studies demonstrated that Cl-IB-MECA inhibited migration of mouse neutrophils isolated from BM towards the chemotactic substance c5a in trans-well migration assays, and inhibited leukocyte migration into the peritoneal cavity in a mouse model of thioglycollate-induced peritonitis. We conclude that treating with the A(3)AR agonist Cl-IB-MECA at the time of reperfusion provides effective protection from ischemia/reperfusion injury in the heart through activation of the A(3)AR expressed in BM-derived cells, potentially by suppressing the robust inflammatory reaction that occurs during reperfusion and neutrophil-mediated tissue injury.

Figure 1

Infarct size (A) and leukocyte accumulation (B and C) induced by 30 min of coronary occlusion and 24 h of reperfusion in B6 wild-type mice and A₃KO mice treated with either vehicle or Cl-IB-MECA throughout the 24 h reperfusion period (100 μg/kg i.v. bolus + 0.3 mg/kg/min by implanting Alzet mini-osmotic pumps). Infarct size is expressed as a percentage of the risk region and leukocyte accumulation determined from histological sections is expressed as the number of leukocytes found within 50 high power fields. n = 8–10 in the infarction studies and 6–8 in the leukocyte accumulation studies. For data shown in A, * indicates P < 0.05 versus the vehicle-treated B6 wild-type group by one-way ANOVA followed by post-hoc contrasts using Student’s t-test for unpaired data with the Bonferroni correction. For data shown in B, * indicates P < 0.05 versus the 24-h time-point in the vehicle-treated group by two-way ANOVA followed by Student’s t-test for unpaired data and the Bonferroni correction.

Figure 2

Reconstitution efficiency of lymphocytes and granulocytes at 2 months after BM transplantation in control studies using CD45-congenic B6 mouse strains as donor (CD45.1⁺)/recipient (CD45.2⁺) pairs. Splenocytes from transplanted recipients were incubated with anti-CD45.1 as a marker for donor cells and either anti-CD11.b (A), anti-GR-1 (B), anti-B22 (C), anti-CD3 (D), anti-CD4 (E), or anti-CD8 (F) antibodies. Labeled cells were analyzed by flow cytometry. The number in each panel indicates the average percentage of cells that were positive for both CD45.1 and each of the other fluorescently marked antibodies from 3 independent experiments.

Figure 3

Effect of treating with Cl-IB-MECA during the reperfusion period on infarct size (A) and leukocyte accumulation (B) in BM-chimeric mice. The infarct size-sparing actions of Cl-IB-MECA were observed in B6→B6 chimeras, but not in A₃KO→B6 chimeras. n = 12–15 in the infarction studies and 8–10 in the leukocyte accumulation studies. Panel C shows the results of quantitative RT-PCR assays measuring A₃AR mRNA expression in spleen tissue randomly chosen from 8 B6→B6 chimeras and 8 A₃KO→B6 chimeras used in the infarction studies. For data shown in Panel A, * indicates P < 0.05 versus the B6→B6 vehicle-treated group by one-way ANOVA followed by Student’s t-test with the Bonferroni correction. For data shown in Panel B, * indicates P < 0.05 versus the vehicle-treated group by Student’s t-test.

Figure 4

Effect of Cl-IB-MECA (100 μg/kg i.v. bolus) on plasma histamine levels (A and B) and systemic blood pressure (C and D) in B6→B6 and A₃KO→B6 chimeric mice. Baseline blood pressure was similar in B6→B6 (85 ± 5) and A₃KO→B6 (89 ± 6) chimeras. *, P < 0.05 versus corresponding values in the vehicle-treated group by 2-way ANOVA followed by Student’s t-test with the Bonferroni correction, n = 6–8/group.

Figure 5

Effect of Cl-IB-MECA on neutrophil migration in trans-well assays using Percoll gradient-purified BM neutrophils. Fluorescently labeled (Calcein-AM) neutrophils were added into upper wells along with either vehicle, Cl-IB-MECA (100 nM), or Cl-IB-MECA (100 nM) in combination with the A₃AR antagonist MRS 5123 (10 μM). The lower wells contained either vehicle or complement component c5a (100 nM). After incubating for 90 min at 37° C, the inserts were removed and fluorescence in each well was quantified. Neutrophil migration was calculated as the percentage of fluorescence found in the wells as a percentage of total fluorescence. Random migration was ~15% in experiments using either wild-type or A₃KO neutrophils. *, P < 0.05 versus the vehicle-treated group by one-way ANOVA followed by Student’s t-test with the Bonferroni correction, n = 6–8/group.

Figure 6

Effect of Cl-IB-MECA on leukocyte accumulation during thioglycollate-induced peritonitis in B6 wild-type mice and A₃KO mice. Mice were injected i.v. with either vehicle or Cl-IB-MECA (100 μg/kg) immediately before intraperitoneal injection with thioglycollate (1 ml of a 3% solution). Three hours later, the number of leukocytes within the peritoneal exudates was quantified. *, P < 0.05 versus the vehicle-treated group by Student’s t-test, n = 8/group.