Abstract 731: Nicotinamide Phosphoribosyltransferase Is A Novel Regulator Of Vascular Smooth Muscle Cell Polarity And Directional Migration
Directional migration of smooth muscle cells (SMCs) is fundamental to the repair and stabilization of atherosclerotic lesions. This form of motility requires a chemotactic source but also depends on the inherent ability of SMCs to crawl in a straight line. We recently discovered that the NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (Nampt; also known as PBEF and Visfatin) was substantially upregulated as human SMCs migrated into patterned, multilayered aggregates (Circ Res, 2005). To determine if Nampt regulates directed SMC motility, human SMCs were infected with retrovirus containing Nampt cDNA and motility parameters quantified by digital time-lapse video microscopy. Compared to vector-infected SMCs, Nampt-overexpressing SMCs displayed a 1.3±0.1-fold increase in migration speed (p<0.05). More striking was the directionally persistent migration path of Nampt-SMCs, with a 2.1±0.2-fold increase in the ratio of net displacement to total distance traveled (D/T, p<0.01). In contrast, inhibition of Nampt activity with FK866 resulted in chaotic motility with lower D/T ratios (p<0.05). Lamellipodial protrusion rate, assessed by rapid acquisition imaging, was increased 2.0-fold in Nampt-overexpressing SMCs (p<0.001). Moreover, the location of the protrusive lamellipod in Nampt-SMCs was restrained to within 15 ± 6o of its original location, but deviated in control SMCs up to 182 ± 56o. This unique anchoring of lamellipodia by Nampt was associated with a 3.2±0.4-fold activation of cdc42, assessed by pull-down of the GTP-bound form. Furthermore, transduction of Nampt-SMCs with dominant-negative cdc42 reversed the lamellipodial anchoring. Finally, using Nampt-GFP-expressing SMCs and FRAP analysis, we established that Nampt underwent microtubule-dependent transported to protrusive lamellipodia.
CONCLUSIONS: Nampt localizes to protruding lamellipodia and imparts SMCs with an increased ability to crawl in a straight line. This innate directionality is mediated by a Nampt-cdc42 axis that restrains the positioning of highly protrusive lamellipodia. These findings identify an internal compass system for SMCs that may be critical for rapid vascular stabilization and repair.