Abstract 3435: GeneChip Analyses Identify Bone Marrow Cells as a Rich Source of Paracrine Factors: Insight From the BOOST Study Program

Abstract

It has been hypothesized that the improvement of left ventricular function observed after intracoronary bone marrow cell (BMC) transfer in patients with STEMI is related in part to paracrine effects. RNA was prepared from BMC that were obtained from patients with STEMI (n=3) included in the ongoing multicenter BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST)-2 trial. Peripheral blood leukocyte (PBL) RNA was obtained from the same 3 patients. GeneChip analyses (Affymetrix HG-U133 Plus 2.0, ca. 47000 probe sets) were performed to characterize the transcriptome in BMC vs. PBL. Using GeneSpring software 7.2, we identified 56 secreted factors that were more strongly expressed (>2-fold) in BMC as compared to PBL. In a second microarray (RNA from n=3 additional patients), 41 of these factors were confirmed. Twelve of these 41 factors were chosen for independent validation by real-time PCR using RNA from 6 additional patients; overexpression in BMC vs. PBL was confirmed for each of these 12 factors. Several factors overexpressed in BMC represented putative cardioprotective cytokines (e.g., BMP-2, CT-1, IGF-1, SDF-1α, VEGF-A). BMC from 7 additional patients were cultured for 24h in serum-free DMEM medium. Conditioned supernatants (SN) were filtered and pooled. BMC-SN (10μL/mL) reduced apoptotic cell death in neonatal rat ventricular cardiomyocytes exposed to simulated ischemia (3h) and reperfusion (1h), as shown by TUNEL (62 ± 7% vs. control, i.e. treatment with unconditioned DMEM medium; n=7; P<0.05) and histone-ELISA (69 ± 12% vs. control; n=5; P<0.05). Moreover, BMC-SN stimulated human umbilical vein endothelial cell (HUVEC) migration (transwell chambers; 293 ± 55% vs. control; n=5; P<0.01) and in-matrigel tube formation (tubular length, 275 ± 35% vs. control; n=5; P<0.05; tube/branch point ratio, 156 ± 10% vs. control; n=5; P<0.05). Finally, BMC-SN promoted enhanced endothelial cell sprouting in the mouse aortic ring assay ex vivo. In conclusion, BMC obtained from patients with STEMI are a rich source of secreted factors. Conditioned BMC supernatants protect cardiomyocytes from I/R-induced apoptosis, and promote pro-angiogenic effects in vitro. Our data tend to support the paracrine hypothesis.