Abstract 18215: Prostacyclin Secretion From Stably Engineered Rat CD45-VEGFR2+ Endothelial Progenitor Cells Preserves Potassium Channel Activity Of Human Smooth Muscle Cells Under Hypoxia
Background: Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis. However, its less than 2 minute half-life makes clinical application cumbersome. Thus, we hypothesized that the use of endothelial progenitor cells (EPCs) which constitutively secrete prostacyclin may overcome this limitation of current prostacyclin therapy.
Methods: We created a cDNA encoding COX-1-10aa-PGIS, linking human cyclooxygenase-1(COX-1) to prostacyclin synthase (PGIS). CD45-VEGFR2+ EPCs were isolated from rat bone marrow mononuclear cells, grown under endothelial conditions, and analyzed by angiogenesis assays. COX-1-10aa-PGIS cDNA was delivered into CD45-VEGFR2+ EPCs by nucleofection. PGI2 secreting single clone derived cell lines (PGI2-EPCs) were established by limiting dilution and G418 selection. Endogenous PGI2 secretion was monitored by ELISA. COX-1-10aa-PGIS protein expression in PGI2-EPCs was identified by western-blot. K+ channel activity was tested by patch clamp techniques on human smooth muscle cells (hSMC) after co-culture with PGI2-EPCs and native EPCs under normal and hypoxic conditions (1.1% O2, 12 hr).
Results: Flow cytometry analysis indicated CD45-VEGFR2+ EPCs expressed CD133, CD31, CD90, and CD29. When seeded on an extracellular matrix, these EPCs exhibited endovascular potential by initiating tube formation after 4 hours. PGI2-EPCs constantly secreted at least ten fold higher levels of endogenous PGI2 in the supernatant as compared to the native EPCs. Western blot analysis confirmed overexpression of COX-1-10aa-PGIS hybrid protein (130 kD) in PGI2-EPCs. Whole-cell patch clamp results revealed that 4-Aminopyridine sensitive K+ current density was increased significantly on hSMCs after co-culture with PGI2-EPCs (12.28 ± 1.69 pA/pF, p<0.05, N=5) compared to native EPCs (6.94 ± 1.73 pA/pF). This enhancement was preserved during hypoxic conditions (10.34 ± 1.51 pA/pF with PGI2-EPCs, vs. 3.88 ± 0.73 pA/pF with EPCs, p<0.01, N=5).
Conclusion: This report shows the first successful establishment of stable PGI2 secreting EPCs. PGI2-EPCs exhibit favorable cell protective effects by efficiently preserving K+ channel activity, an indication of hyperpolarization and relaxation of hSMCs under hypoxia.
- © 2010 by American Heart Association, Inc.