Activation of neuronal nitric oxide synthase (nNOS) mediates proneurotrophin (proNT)‐induced apoptosis of smooth muscle cells (SMCs)

Apoptosis of SMCs is a well‐characterized component of the pathogenesis of atherosclerosis. We identified the p75 neurotrophin receptor (p75) as a regulator of SMC apoptosis in vascular lesions. p75 and its ligands, the proNT, are expressed in atherosclerotic lesions and pro nerve growth factor (pNGF) induces apoptosis of p75‐SMCs. Moreover, there is reduced apoptosis and increased lesion development in the ligated carotid artery of p75−/−. The signaling pathways mediating p75‐induced apoptosis, however, remain unclear. We now demonstrate that activation of nNOS mediates p75‐induced apoptosis of SMCs. The NOS inhibitor, L‐NMA, dose‐dependently inhibits pNGF‐induced, but not FAS‐induced, apoptosis of SMCs. Maximal inhibition of pNGF‐apoptosis is 75% with 1.2 mM L‐NMA. D‐NMA, the inactive isoform, is without effect. Western blot analysis and RT‐PCR show nNOS to be the NOS isoform predominately expressed by SMCs. Double immunofluorescence and cell fractionation studies indicate that in SMCs, nNOS localizes to mitochondria. Immunofluorescence analysis for nitrotyrosine residues (NT), an index of NOS activation, demonstrates that pNGF increases NT levels by 2–3 fold. In addition, neointimal SMCs in human and murine atherosclerotic lesions express p75NTR and nNOS. Finally, pNGF activates p38 and JNK, kinases implicated in mitochondrial‐dependent apoptosis, and p38 inhibitors reduce pNGF‐induced apoptosis. These data are the first to identify ligand‐dependent activation of mitochondrial nNOS in SMC and characterize a novel signaling pathway mediating p75‐induced apoptosis. These studies were supported by NIH RO1HL‐58623.