Abstract 1030: Interleukin-1β Downregulates the Expression of Vascular Endothelial Growth Factor-D (VEGF-D) and Inhibits Angiogenesis in Cardiac Microvascular Endothelial Cells (CMEC’s)
Angiogenesis is essential in the repair of the heart following myocardial infarction. Levels of proinflammatory cytokine, interleukin-1β (IL-1β), increase in the heart post-MI. Here we investigated the role of IL-1β in the regulation of VEGF-D expression and tested the hypothesis that IL-1β inhibits angiogenesis in CMECs.
Methods and Results: Primary cultures of CMECs, isolated from adult rat hearts, were exposed to IL-1β (4ng/ml) for 24h. Differential expression of angiogenic genes, using SuperArray analysis, indicated that IL-1β downregulates VEGF-D expression. RT-PCR confirmed a decrease of 67±9% in VEGF-D mRNA levels (p<0.05 vs. control; n=3). Western blot analyses demonstrated a 60±7% (p<0.05 vs. control; n=6) decrease in VEGF-D protein following IL-1β stimulation. Inhibition of PKCα/β1, ERK1/2, or JNKs using Gö 6976 (100nM), UO126 (10 μM), or SP600125 (10 μM) significantly prevented IL-1β-stimulated decreases in VEGF-D protein levels. MnTMPyP (10 μM, superoxide dismutase mimetic) and SN50 (10 μM; inhibitor of NF-κB) exhibited synergistic effects with IL-1β, further decreasing VEGF-D protein levels by 81±6% and 89±3% respectively (p<0.05 vs. IL-1β; n=3–5). Wound healing assays indicated that IL-1β promotes cell migration, decreasing the wound distance by 82 μm after 24h (p<0.05 vs. time 0; n=4) while there was no significant difference in control cells. However, IL-1β inhibited cell-to-cell contact formation within the wound while treatment with VEGF-D promoted migration and complete healing of the wound within 24h. Phalloidin-FITC staining demonstrated that IL-1β significantly reduces actin polymerization (p<0.01 vs. control; n=8), a pre-requisite for tube formation. Tube formation, analyzed using 3-D collagen gels, was also inhibited by IL-1β treatment.
IL-1β downregulates VEGF-D expression via the activation of PKCα/β1, ERK1/2, and JNKs,
production of reactive oxygen species and activation NF-κB signaling pathways inhibit IL-1β-stimulated decreases in VEGF-D expression, and
IL-1β inhibits cell-to-cell contact formation, actin polymerization, and tubular structure formation.
Thus, IL-1β may inhibit angiogenesis in cardiac endothelial cells, possibly via the inhibition of VEGF-D.